Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25

Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25 Gamze Balcı, Esra Ayan, Ahmet Katı This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6555765/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial peptides (AMPs) are natural compounds with broad-spectrum activity, playing a key role in the innate immune system by disrupting pathogen membranes. This study evaluates the recombinant antimicrobial peptide GFP-Hepc25, where GFP tagging facilitates fluorescence monitoring without additional staining. Optimal growth conditions for GFP-Hepc25 expression were determined as 18°C, IPTG concentrations of 0.1–1 mM, and a casein-based medium with yeast extract and NaCl. Both GFP-Hepc25 and Hepc25 demonstrated antimicrobial activity against E. coli and S. aureus . GFP-Hepc25 exhibited notable stability under heat, and acidic conditions, as well as in the presence of MnCl₂, ZnSO₄, MgSO₄, CuSO₄, and FeSO₄. It retained stability at 100°C for 5 and 10 minutes, though prolonged heating caused degradation. However, stability decreased under alkaline pH and with detergents such as SDS, Triton-X100, and Tween20. GFP-Hepc25 significantly inhibited S. aureus and P. aeruginosa biofilm formation. These findings highlight GFP-Hepc25 as a promising next-generation antimicrobial peptide. Antimicrobial peptide GFP-Hepc25 Hepc25 Fermentation optimization Peptide Stability Minimum inhibitory Concentration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Hepcidin was discovered as the liver-expressed antimicrobial peptide in 2000 (Krause et al. 2000). Hepcidin is encoded by the hepcidin antimicrobial peptide ( HAMP ) gene. It is first produced as an 84-amino acid pre-pro-hepcidin, which is then processed into a 60-amino acid pro-hepcidin. Finally, it is sliced to a mature C-terminal 25 amino acid active peptide (Rochette et al. 2015). Hepcidin is a crucial regulator of iron homeostasis in the body, mainly by controlling the expression of ferroportin-1 (FPN-1), an iron-exporting protein found on duodenal enterocytes and macrophages (Ganz and Nemeth 2012). Hepcidin-25 (Hepc25), one of its major and active isoforms, plays a critical role in both iron metabolism and innate immunity. Structurally, it consists of 25 amino acids and possesses a distinctive β-hairpin loop stabilized by four disulfide bonds, which contributes to its biological activity (Rauf et al. 2020). The principal function of Hepc25 is to regulate iron levels by binding to the iron exporter ferroportin, found on the surface of enterocytes, macrophages, and hepatocytes. Once bound, Hepc25 induces the internalization and degradation of ferroportin, reducing the release of iron into circulation. This process is crucial in preventing excessive iron accumulation, which could otherwise catalyze the formation of harmful free radicals through the Fenton reaction (Winterbourn 1995). Hepc25 expression is modulated by factors such as inflammation, hypoxia, and iron levels. During infection or inflammation, cytokines like IL-6 upregulate Hepc25 expression, leading to iron sequestration (a mechanism known as the anemia of inflammation). Consequently, dysregulation of Hepc25 can result in iron-related disorders such as anemia of chronic disease or iron overload conditions like hereditary hemochromatosis (Arezes and Nemeth 2015). Beyond its role in iron regulation, Hepc25 exhibits antimicrobial properties. By limiting iron availability, it creates a hostile environment for pathogens that require iron for survival and growth. Additionally, Hepc25 has recently gained attention as a potential antimicrobial agent with therapeutic applications for treating various infectious diseases; it is directly bactericidal activity against several microorganisms, reinforcing its role in the innate immune response (Ganz 2011). Human Hepc25 was successfully expressed recombinantly in Escherichia coli . However, since the bacterial cytosol cannot form and maintain disulfide bonds, an additional in vitro refolding step was necessary to fold the peptide properly (Zhang et al. 2005; Achmüller et al. 2007). To address this challenge, the E. coli Origami strain was employed (Gagliardo et al. 2008). The bacterial expression system is straightforward, requires minimal time for DNA manipulation, and typically achieves high protein expression levels. While the E. coli expression system offers several advantages, it also comes with notable limitations. These include challenges like codon usage bias, proteolytic degradation of the expressed protein, difficulties in protein release, and potential toxicity of the expressed peptides to the host cells. Notably, non-fusion Hepc25 is often produced as inclusion bodies, necessitating complex and time-intensive refolding steps to recover appropriately folded protein structures (Zhang et al., 2005). Various protein fusion technologies have been developed to enhance the expression of target peptides in host cells, including glutathione-S-transferase, small ubiquitin-related modifier protein, and thioredoxin gene fusion systems (Sadr et al. 2017). Despite their utility, these systems may still face challenges, such as inconsistent expression levels and reduced efficiency (Zuo et al. 2005). Otherwise, green fluorescent protein (GFP) appears as a unique protein marker due to its two key advantages that (i) fluorescence of GFP without the need for additional co-factors or staining, and (ii) fluorescence of GFP is easily observable from outside the cells (Chalfie et al. 1994). Initially discovered in the jellyfish Aequorea victoria , GFP was first identified and described in 1962 (Shimomura et al. 1962), and its cDNA was successfully cloned in 1992 (Prasher et al. 1992). Recently, GFP fusion technology has been applied to enhance protein production in E. coli . Fusing GFP to the N-terminus of target proteins improves protein solubility, expression levels, and purification efficiency, making valuable tool of GFP use for recombinant protein expression (Cha et al. 2000). Earlier studies have highlighted using GFP as a quantitative marker for protein expression in insect cell systems (Cha et al. 1999) and insect larvae (Cha et al. 1997, Cha et al. 1999). Additionally, research demonstrated its utility in E. coli fermentation processes, where GFP and the target protein, chloramphenicol acetyltransferase (CAT), were co-expressed in an operon system with identical ribosome binding sites for both genes ( GFP and cat ) (Albano et al. 1998). A study conducted in 1999 further validated GFP as a noninvasive, quantitative fusion marker for monitoring foreign protein production in recombinant E. coli (Cha et al. 2000). In a study, a GFP-hepcidin chimera was created by fusing GFP to the C-terminus of hepcidin, and the resulting construct was expressed in Huh7 eukaryotic cells (Chachami et al. 2013). Stable, low-level expression facilitated the secretion of correctly matured hepcidin-GFP. The secreted chimera was demonstrated to be active in regulating iron homeostasis. Under hypoxic conditions, the cellular localization of pro-hepcidin-GFP was altered, and the secretion of mature hepcidin-GFP was significantly reduced. To the best of our knowledge, the potential of GFP fusion technology for producing recombinant human Hepc25 has not been thoroughly explored. Thus, this study aimed to establish a robust method for producing soluble, functional recombinant human Hepc25 by employing codon optimization and a novel GFP-based strategy in an E. coli expression system. Following purification, the peptide's characteristics were evaluated using various assays to confirm its functionality. Methods Materials, and Bacteria Strains The strains E. coli Rosetta (DE3), Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, P. aeruginosa ATCC9027 were obtained from the University of Health Sciences. Bacteria were cultured in LB (Luria-Bertani broth Lennox, BD Difco, Canada) at 37◦C. Pierce™ BCA Protein Assay Kit, and HisPur™ Ni-NTA Resin was purchased from Thermo Scientific (Thermo Scientific™, USA). The designed plasmid is provided by Twist Bioscience (twistbioscience.com, South San Francisco). Construction of the fusion expression plasmid pET28a(+)-GFP-Hepc25 The antimicrobial peptide was designed using SnapGene, followed by recombinant production. The genes encoding the protein were cloned into the pET28(a) vector with a 6-histidine tag fusion through a service provided by Twist Bioscience (twistbioscience.com). The plasmid's gene sequence consists of the 6-histidine tag, green fluorescent protein, TEV fusion protein, and the Hepc25 sequence (Figures S1 and S2). The plasmid was then transformed into the E. coli Rosetta (DE3) for expression testing. Based on the peptide sequence, GFP-Hepc25 was determined to be approximately 34 kDa, while Hepc25 was 6 kDa. Table 1 Hepcidin25 Amino Acid Sequence Hepcidin25 Amino Acid Sequence DTHFPICIFCCGCCHRSKCGMCCKT Preparation of Test Expression The GFP-Hepc25, ordered in a lyophilized form, was reconstituted by adding 1,000 µl of RNase and DNase-free water. The GFP-Hepc25 was then successfully transformed into a competent E. coli Rosetta (DE3) following the Durdagi et al. study (Durdagi et al. 2021 ). Briefly, the GFP-Hepc25 and E. coli Rosetta (DE3) mixture was incubated on ice for 20 minutes. In a sterile Eppendorf tube, 15 µl of bacterial cells and 3 µl of plasmid DNA were combined, followed by an additional incubation on ice for 30 minutes. To facilitate the transformation, the mixture underwent a heat shock at 42°C for 30 seconds, immediately followed by cooling on ice for 5 minutes. Subsequently, 400 µl of LB broth medium (g/L Tryptone 10.0, Yeast Extract 5.0, Sodium Chloride 5.0) was added to the tube, and the suspension was incubated at 37°C for 1 hour. The bacterial culture was then plated onto LB agar (g/L Tryptone 10.0, Yeast Extract 5.0, Sodium Chloride 5.0), Agar 10.0) containing kanamycin (50 ug/mL) and incubated at 37°C for 16 to 24 hours. The next day, the transformed colonies were cultured in an LB broth medium with kanamycin (50 ug/mL) to ensure selection. Glycerol stocks of the successfully transformed colonies were prepared for long-term storage. Optimization of Growth Media by Micro-scale Fermentation In order to determine the optimal production conditions during fermentation, 11 different media were used (Table 2 ). The optimization conducted using the BioLector microbioreactor (Beckman Coulter, BiolectorPro, USA) involved the following media and their compositions: Table 2 The carbon, nitrogen, and mineral source components of the medium used for optimization Carbon Source Nitrogen Source Minerals Ingredients (g/L) /Number Casein Peptone Glucose Tryptone Dekstrose Gelatin Yeast Extract Beef Extract Papain digest of soybean NaCl NaCl & Dipotassium Phosphate 1 10,0 - - - - - 5,0 - - 5,0 - 2 10,0 - - - - - 5,0 - - 10,0 - 3 - 10,0 - - - - 5,0 - - 5,0 - 4 - 10,0 - - - - 5,0 - - 10,0 - 5 - - 10,0 - - - 5,0 - - 5,0 - 6 - - 10,0 - - - 5,0 - - 10,0 - 7 - - - 10,0 - - 5,0 - - 5,0 - 8 - - - 10,0 - - 5,0 - - 10,0 - 9 - - - 15,0 - - 7,5 - - 7,5 - 10 - - - - - 5,0 - 3,0 - - - 11 17,0 - 2,5 - - - - - 3,0 - 5,0 & 2,5 These media were selected to examine the effects of carbon and nitrogen sources, as well as the impact of NaCl concentration (ionic environment) on GFP-Hepc25 production. The first eight media were prepared to compare the effect of different nitrogen sources (casein, peptone, glucose, tryptone) with yeast extract as a constant carbon source. The effect of varying NaCl concentrations was also evaluated. Additionally, formulations containing beef extract, Papain digest of soybean, and dipotassium phosphate were incorporated into the study to evaluate their effects on microbial growth and GFP-Hepc25 production. In this study, medium No. 7 (LB - Luria-Bertani broth), medium No. 10 (NB - Nutrient broth), and medium No. 11 (TSB - Tryptic soy broth) were commercially procured. The Rosetta strain was inoculated into kanamycin-containing media. The culture was incubated in a shaking incubator at 37°C and 150 rpm for 1–2 hours. When the optical density at 600 nm (OD 600 nm) reached approximately 0.4, induction was performed with 0.4 mM IPTG. Subsequently, the cultures were incubated in a bioreactor at 18°C with agitation at 800 rpm for 24 hours. Following incubation, the collected samples were centrifuged at 4,000 × g for 30 minutes. The pellet was then resuspended in 200 µl of Phosphate-Buffered Saline (PBS). The collected samples were analyzed by electrophoresis on a 15% SDS-PAGE gel. Optimization of IPTG Concentration with Incubation Time The E. coli Rosetta (DE3) was inoculated into a medium-II (optimized media, scenario 2, containing Casein 10 g/L, Yeast Extract 10 g/L, NaCl 10 g/L) with kanamycin. The culture was incubated in a shaking incubator at 37°C and 150 rpm for 16–24 hours. In the fermentation cultures prepared in the microbioreactor, the final IPTG concentrations were adjusted to 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 1.0 mM. The cultures were then incubated at 18°C and 800 rpm for 20 hours. The protein production was monitored over a 20-hour period. The positive control consisted of a bacteria-free medium-II, while the negative control was a bacterial strain not induced with IPTG. Optimization of Incubation Temperature After Induction To optimize GFP-Hepc25 production, fermentation cultures (100 mL each, prepared in Erlenmeyer flasks) were incubated at different temperatures to assess the impact of temperature on production yield. Cultures reaching an OD 600 nm value of approximately 0.4 were induced with 0.1 mM IPTG and then incubated at 18°C, 30°C, and 37°C in a shaking incubator for 24 hours. After incubation, samples were collected and centrifuged at 4000 xg for 30 minutes. The fluorescence emission intensity of the samples was measured using a Multimode Reader (Agilent Technologies, BioTek Synergy Neo2, USA). The fluorescence emission intensities were compared, utilizing the green fluorescent protein, to determine the optimal conditions for production. Samples were taken before and after centrifugation, as well as from the supernatant and pellet fractions, followed by 15% SDS. The thickness of SDS-PAGE bands was visually distinguishable, allowing for a clear comparison of protein expression levels between samples, leading to the selection of the optimum incubation temperature. Large-scale Fermentation Culture Bacteria that include GFP-Hepc25 plasmid were inoculated into 300 mL of media-II supplemented with kanamycin (50 ug/mL). After incubation at 37°C with shaking at 150 rpm for 16–24 hours, 250 mL of this pre-culture was transferred into 5 L of bioreactor (500 rpm, 37°C) medium with kanamycin (50 ug/mL). Once the OD 600 nm reached 0.4, the culture was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The fermentation was carried out in a shaking incubator at 18°C and 250 rpm for 16–24 hours. Protein Purification Following fermentation, the peptides present in the culture were purified. The samples were first centrifuged at 4,000 × g for 30 minutes. After discarding the supernatant, the pellet was prepared for subsequent processing steps. A lysis buffer was prepared containing 500 mM NaCl, 50 mM Tris-HCl, 20 mM imidazole, 5% glycerol, and 0.4% Triton X-100. This buffer was stored at + 4°C, with 5 mM β-mercaptoethanol (BME) added immediately prior to use. The pellets were resuspended in 45 mL of lysis buffer and subjected to three rounds of sonication for 30 seconds each at 70% power while maintained on ice. The lysate was then centrifuged at 10,000 rpm for 30 minutes. The resulting supernatant was purified using nickel-NTA affinity chromatography via Fast Protein Liquid Chromatography (FPLC). The purification process involved sequential application of equilibration buffer (25 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0), sample loading, and wash buffer (25 mM Tris-HCl, 150 mM NaCl, 40 mM imidazole, pH 8.0), followed by elution with elution buffer (25 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0) into sterile collection tubes. The column was subsequently regenerated using 0.5 M NaOH followed by 20% ethanol and stored at + 4°C. For downstream applications, the isolated peptides were dialyzed against dialysis buffer (25 mM Tris-HCl, 150 mM NaCl) using a 10 kDa molecular weight cut-off membrane to remove imidazole, with overnight incubation at + 4°C under constant stirring. Finally, the purified peptides were supplemented with 10% glycerol, aliquoted, and stored at -80°C for long-term preservation. Antimicrobial Activity Assay of GFP-Hepc25 and Hepc25 The antimicrobial activity of GFP-Hepc25 and Hepc25 against E. coli and S. aureus was determined using the Minimum Inhibitory Concentration (MIC) assay (Wiegand et al. 2008 ). The concentration of GFP-Hepc25 and Hepc25 were calculated using the BCA Protein Assay kit. In the MIC test, 50 µL of Mueller Hinton Broth medium (MHB) (containing per liter: 2.0 g beef infusion solids, 17.5 g casein hydrolysate, and 1.5 g starch) was added to each well of a 96-well plate. An initial 100 µL aliquot of the antimicrobial peptide (AMP) was added to the first column and subjected to two-fold serial dilution up to the 10th column. Subsequently, 50 µL of microbial suspension, standardized to 0.5 McFarland (1×10⁸ CFU/mL), was inoculated into each well. Following 24-hour incubation at 37°C, the OD 600 nm was measured using a spectrophotometer. The negative control consisted of microorganisms without GFP-Hepc25 or Hepc25, while the positive control contained only MHB medium. This experimental setup allowed for accurate determination of the minimum peptide concentrations required to inhibit visible growth of the test microorganisms. Stability Analysis of GFP-Hepc25 in Selective Heat Treatment The stability of the peptide was examined after exposure to 100°C for different time intervals. In an Eppendorf tube, 1 mL of the GFP-Hepc25 (800,0 µg/mL) was subjected to 100°C for 5, 10, and 30 minutes using a heat block (Fahimirad et al. 2021 ). As a control, the peptide was kept at 37°C. After heating, the peptides were placed on ice and subjected to MIC testing against E. coli and S. aureus . The MIC test is performed by preparing serial dilutions of the GFP-Hepc25 in a growth medium, inoculating each with the test microorganism, and incubating under suitable conditions. The MIC is the lowest concentration of GFP-Hepc25 that prevents visible microbial growth. Stability Analysis of GFP-Hepc25 in Selective Heavy Metal Treatment The stability of the peptide against various metal salts was assessed by incubating the peptide with the salts at 37°C for 1 hour. For this purpose, MgSO₄, FeSO₄, MnCl₂, ZnSO₄, and CuSO₄ were used (Choyam et al. 2021 ), with each metal salt adjusted to a final concentration of 1.0 mg/mL. The peptides were mixed with the metal salts in Eppendorf tubes and maintained at a constant temperature for the specified duration. As a control, the peptide was incubated under the same conditions without exposure to the metal salts. After incubation, the antimicrobial activity of the peptides was evaluated using the MIC test on E. coli and S. aureus as indicator microorganisms. Stability Analysis of GFP-Hepc25 in Selective Chemical Treatment Solutions of sodium chloride, acetic acid, ascorbic acid, benzoic acid, sodium benzoate, and sodium sulfite were prepared at different concentrations (Choyam et al. 2021 ). Each (1 mg) preservative was mixed with the (1 mg/mL) GFP-Hepc25 and incubated at 37°C for a minimum of 2 hours. As a control group, the GFP-Hepc25 was incubated under the same conditions without exposure to any food preservatives. After incubation, the antimicrobial activities of the peptides were measured using the MIC test on indicator microorganisms such as E. coli and S. aureus . Stability Analysis of GFP-Hepc25 in Selective Detergent Treatment The stability of the peptide against various detergents and solvents was evaluated by incubating it at 37°C for 5 hours (Choyam et al. 2021 ). For this purpose, solutions containing 1% sodium dodecyl sulfate (SDS), Tween 80, and Triton X-100 were prepared. Each solution was mixed with a 1 mg/mL peptide sample and incubated under the appropriate conditions. As a control group, the peptide was incubated at the same temperature without exposure to any detergents or solvents. After the incubation period, the antimicrobial activity of the peptides was assessed using the MIC test on indicator microorganisms such as E. coli and S. aureus . Stability Analysis of GFP-Hepc25 in Selective pH Treatment The stability of the peptide (800.0 µg/mL) under different pH conditions was evaluated by incubation at 37°C for 2 hours (69). For this purpose, peptide solutions were adjusted to pH 5, 7, 9, and 13, along with a control group, and incubated at 50 rpm. Following incubation, all samples were readjusted to pH 8. As a control, the peptide was incubated without any pH modification. After the incubation period, the antimicrobial activity of the peptides was assessed against microorganisms including E. coli and S. aureus using the MIC test. This experimental design allowed for systematic evaluation of pH-dependent stability while maintaining consistent testing conditions through pH readjustment prior to antimicrobial assessment. Growth Inhibition Assay of GFP-Hepc25 The growth inhibition of E. coli and S. aureus treated with GFP-Hepc25 was assessed by monitoring the absorbance at OD 600 nm at multiple time points. The strains were cultured in 96-well plates under conditions similar to those used for the determination of the MIC test. Various concentrations of GFP-Hepc25 were tested, and the bacterial growth was measured at every 5 hours (0, 5, 10, 15, 20, and 24) hours. The absorbance at OD 600 nm was recorded using a spectrophotometer. The differences in absorbance at each time point were used to determine the antimicrobial effect, revealing the dynamics of bacterial growth inhibition in response to GFP-Hepc25 (Luo et al. 2022 ). Morphological Alteration of Microorganisms The E. coli pre-culture was sub-cultured into a fresh LB broth medium. When the OD 600 nm reached 0.6, it was diluted 1:100 (Zhao et al. 2019 ). In a sterile Falcon tube, 1 mL of 10⁶ CFU/mL bacterial culture was treated with 1 mL LB broth and 1 mL of GFP-Hepc25 (800.0 µg/mL) and incubated at 150 rpm, 37°C for 2 hours. In the control group, PBS was added instead of GFP-Hepc25. The samples were then centrifuged at 3000 xg for 5 minutes to pellet the bacteria and washed with PBS. Next, a 2.5% glutaraldehyde solution was added, and the samples were incubated overnight at + 4°C. The bacteria were washed with 0.1% PBS and subjected to serial ethanol washes. Each wash step was performed for 10 minutes. Ethanol was applied in increasing concentrations: 30%, 50%, 70%, 90%, 100%, and then again 100% in order to progressively dehydrate the samples and prepare them for electron microscopy analysis. Finally, the samples were placed on sticky carbon tape, dried, and observed under a scanning electron microscope (SEM). Biofilm Formation Assay The biofilm inhibition test is an analytical method used to evaluate the degree to which an agent or condition prevents microorganisms from forming biofilms on surfaces ( O'Toole 2011 ). Staphylococcus aureus and Pseudomonas aeruginosa are pathogenic bacteria with strong biofilm-forming capabilities and are commonly used in biofilm inhibition tests as they represent both Gram-positive and Gram-negative strains. The biofilm inhibition activity of GFP-Hepc25 peptide against S. aureus and P. aeruginosa was determined using the crystal violet method in 96-well plates. S. aureus was incubated in Tryptic Soy Broth (TSB) medium (g/L: 17 g pancreatic casein, 2.5 g glucose, 3 g papaic soybean, 2.5 g dextrose, 5 g sodium chloride, 2.5 g dipotassium phosphate) with 0.2% glucose addition at 37°C for 16–24 hours. P. aeruginosa was incubated in Nutrient-Rich LB Broth (g/L: 15.0 g tryptone, 7.5 g yeast extract, 7.5 g sodium chloride (NaCl) and then diluted 1:100 in fresh medium. In 96-well plates, 160 µL of microbial suspension and 20 µL of peptide solution at different concentrations were added. For the negative control, 160 µL of microorganism and 20 µL of medium were used, while the positive control consisted only of medium without microorganisms. Additionally, controls containing 50% and 10% bleach were tested along with the peptides, as bleach prevents biofilm formation. After 24-hour incubation at 37°C, the wells were first washed with PBS, then fixed with methanol and stained with 0.1% crystal violet for 30 minutes. The wells were then washed with sterile water and 200 µL of 95% ethanol was added. Absorbance was measured at OD 570 nm. Biofilms, which are sticky structures formed by microorganisms on surfaces, were analyzed by staining with crystal violet to assess biofilm presence. Cytotoxicity Test The L929 cell line was obtained from the American Type Culture Collection (NCTC clone 929: CCL-1). These cell lines were cultured and expanded in cell growth medium containing 10% Fetal Calf Serum (FCS), 1.0% penicillin/streptomycin (P/S), and 1% GlutaMAX. Cells were detached using 0.25% EDTA/trypsin and pelleted by centrifugation at 1,200 rpm for 4 minutes. After resuspension in fresh cell growth medium, the cells were passaged by seeding onto new cell culture plates. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) and grown in a cell culture incubator at 37°C with 5% CO₂ atmosphere. These prepared cells were then subjected to various experimental procedures involving specified samples and reagents for conducting cytotoxicity tests. MTT Assay Cell viability was assessed using the MTT assay (Riss et al. 2013 ). L929 cells were seeded in a 96-well plate at a density of 10,000 cells per well and incubated for 24 hours at 37°C with 5% CO₂. Following incubation, the cell culture medium was replaced with fresh medium containing varying concentrations of the test compound. After exposure periods of 24, 48, and 72 hours, the test medium was aspirated, and 110 µL of MTT solution (5.0 mg/mL in PBS, 10% v/v) was added to each well, followed by a 4-hour incubation. The formazan crystals were solubilized by adding 100 µL of SDS solution (1% w/v in 0.01 M PBS, pH 7.4) to each well, with an additional 24-hour incubation period. The absorbance of the formazan product was then measured at OD 570 nm and OD 630 nm, and the results were calculated and compared to the control group. Statistical analysis The data were analyzed using statistical methods based on results from at least three independent experiments, and the findings were expressed as mean ± standard deviation (SD). Student’s t-test and Linear Regression were applied to assess differences between groups. Results with a p-value < 0.05 were considered statistically significant. Additionally, to provide a precise analysis of antimicrobial activity, MIC (Minimum Inhibitory Concentration) values were determined using the Gompertz model. Results and Discussion GFP-Hepc25 and Hepc25 alone have matching performance and antimicrobial potency GFP-Hepc25 has demonstrated significant antimicrobial activity in performance studies conducted so far. To determine if this activity originates from the GFP tag or the Hepc25 itself, both GFP-fused and Hepc25-alone peptides were subjected to antimicrobial activity assays (Fig. 1 ). Accordingly, the antimicrobial activity of GFP-Hepc25 and Hepc25 alone were confirmed against both E. coli and S. aureus . Calculations revealed that the MIC of GFP-Hepc25 against E. coli was 258.264 µg/mL, while the MIC against S. aureus was 377.74 µg/mL (Fig. 1 A-B). Otherwise, the MIC of cut Hepc25 against E. coli was 279.84 µg/mL, while the MIC against S. aureus was 238.74 µg/mL (Fig. 1 C-D). These MIC values represent the minimum concentration of hepcidin required to effectively inhibit microbial growth. The calculated MIC values are provided in Fig. 6 in detail. There is no significant difference in antimicrobial activity between GFP-Hepc25 and Hepc25 against E. coli . However, for S. aureus , the MIC value of the GFP-Hepc25 is 377.74 µg/mL, while the MIC for the non-GFP-tagged peptide is 238.74 µg/mL. The higher MIC for the GFP-Hepc25 suggests lower antimicrobial activity compared to the Hepc25. Conversely, the lower MIC for Hepc25 indicates greater antimicrobial efficacy. Although the difference in MIC values between the two forms of hepcidin for S. aureus is relatively small, these results suggest that GFP itself does not exhibit toxic effects, which is in line with the literature knowledge related to its active use for gene expression in mammalian cells of GFP (Alexander et al. 1997 ). This observation supports the conclusion that GFP tagging does not compromise the antimicrobial potential of the hepcidin peptide; instead stands out as a highly efficient step in the solubilization of hepcidin and easy recombinant production and purification process (Rücker et al. 2001 ;Waldo et al. 1999 ). Here, we propose highly yielded and cost-effective recombinant GFP-Hepc25 complex production and purification path. Well-known that recovery of recombinant proteins from bacterial systems often demands meticulous fine-tuning of expression and purification conditions. To significantly simplify this process, GFP was integrated into bacterial expression vectors. GFP not only improves folding efficiency and solubility (Rücker et al. 2001 ; Alfasi et al. 2011 ) but also streamlines production and purification workflows through highly refined methodologies. Moreover, this fusion strategy facilitates the precise detection of GFP-tagged hybrid proteins in intact bacterial cells via fluorescence microscopy (Venkatesh et al. 2005 ). To efficiently evaluate a range of conditions critical for protein expression, GFP fluorescence, which reflects expression levels, was directly measured in live bacterial cultures using a fluorescence plate reader (Chachami et al. 2013 ). This approach enables real-time monitoring of protein expression (Venkatesh et al. 2005 ) (Fig. S4 and S6) and suggests that GFP tagging enhances protein stability and solubility (Rücker et al. 2001 ) (Fig S5 and S7, and S8) instead traditional approach (Zhang et al. 2005 ). Furthermore, our GFP-fused Hepc25 exhibits antimicrobial activity comparable to or exceeding that of Hepc25 alone (Fig. 1 ), aligning with findings from numerous studies (Maisetta et al. 2010 ; Lombardi et al. 2015 ). Notably, Hepc25 demonstrates greater antimicrobial activity at acidic pH (Fig. 4 ) (Maisetta et al. 2010 ; Maisetta et al. 2013 ), stressing the potential of GFP-Hepc25 as a strategic framework for developing advanced therapeutics aimed at infections localized in physiologically acidic environments. Integrating the GFP tag enables precise real-time monitoring, further enhancing its applicability. This approach positions engineered Hepc25-derived peptides as a cutting-edge solution for combating a broad spectrum of infections across various anatomical niches and pathological conditions. However, challenges such as the complex synthesis required to establish disulfide bridges critical for antimicrobial activity (Shike et al. 2002 ), partial inhibition by body fluids (Maisetta et al. 2008 ), and the relatively high concentrations needed for activity pose significant barriers to the clinical application of hepcidin-derived peptides. These limitations underscore the necessity of further experimental research to optimize these peptides for therapeutic use. Alternatively, a more cost-effective and efficient approach may involve the recombinant production of Hepc25 fused with a GFP tag, presenting promising opportunities for developing a novel GFP-Hepc25 complex as a potential antimicrobial peptide, maybe offering an innovative alternative to next-generation antibiotics. Selective optimizations of recombinant GFP-Hepc25 made cost-efficient paradigm Once the successful transformation of the GFP-Hepc25 sequence that was cloned into the pET28(a) vector, test expression revealed substantial protein accumulation in the cytosol of the host cells (Fig. S3). These results are consistent with findings reported in the literature (Sadr et al. 2017 ). Initially, media optimization was conducted to identify the most efficient medium for experimental procedures. The experiment was performed on a microscale using the BioLector microbioreactor. The media were ranked according to fluorescence intensity measured in a bioreactor, from highest to lowest, nu: 2 > 1 > 11 > 3 > 4 > 6 > 8 > 9 > 7 > 5 > 10. This ranking indicates that the second composition (Casein 10 g/L, Yeast Extract 10 g/L, NaCl 10 g/L) yielded the highest fluorescence intensity, reflecting the most efficient Hepc25 production, while other media showed progressively lower performance (Fig. S4). The fluorescent capability of GFP (Chachami et al. 2013 ) allowed real-time monitoring of protein intensity during BioLector screening, offering that GFP-tagged protein production is not only advantageous for improving protein solubility but also serves as a valuable method for real-time tracking and analysis. This is in line with our SDS-PAGE result (Fig. S5). Once the max media optimization, cells containing the GFP-Hepc25 complex were screened at the micro-scale with different IPTG values (0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 1.0 mM) to find the most efficient induction value (Fig. S6). Accordingly, at the end of the incubation in the BioLector microbioreactor, bacterial growth curves and fluorescence measurements at 436/540 nm (excitation/emission) wavelengths were analyzed to assess GFP-tag expression. The bacterial growth curve showed an increase in growth across all samples except for the positive control The positive control consisted of a bacteria-free medium, while the negative control was a bacterial strain not induced with IPTG. The GFP-tag expression graph indicated an increased trend in fluorescence for all samples, excluding the positive and negative controls. The time-dependent growth curve of the E. coli Rosetta (DE3) was monitored using a BioLector. By analyzing the time-dependent fluorescence measurements at 436/540 nm (excitation/emission) wavelengths, it was observed that induction with varying concentrations of IPTG successfully enhanced the protein's expression levels. Induction was successful at all IPTG concentrations, with increasing fluorescence intensity indicating a corresponding rise in GFP-Hepc25 expression. In contrast, no increase in expression was observed in the negative control, which was not induced with IPTG. The results demonstrate effective induction and protein expression across all IPTG concentrations tested, ranging from 0.1 mM to 1 mM. This agrees with our SDS-PAGE analysis (Fig. S7). The thickness and intensity of the bands in the gel correspond to the protein expression levels, which increased with IPTG concentration. In the negative control, which was not induced with IPTG, no discernible band was observed. These findings confirm, consistent with the fluorescence analysis, that protein expression was successfully induced in a concentration-dependent manner. Based on this result and literature, we prefer to use 0.1 mM IPTG amount in further experimental setup to provide cost-effective recombinant production ( Silaban 2018 ). Reducing the concentration of IPTG, a widely used inducer in recombinant protein production, offers significant economic and industrial advantages (Larentis et al. 2014 ). IPTG is a costly chemical, and its reduced usage can substantially lower production expenses, particularly in large-scale industrial applications. Utilizing low IPTG concentrations while maintaining efficient protein yields minimizes process costs, making production more economically viable (Lecina et al. 2013 ). Additionally, lower IPTG levels help reduce cell stress, ensuring better cell health and consistent protein production, which is crucial for industrial-scale operations (Olaofe et al. 2010 ;Lecina et al. 2013 ). Post-induction temperature is a critical parameter in proteins with IPTG added since the temperature following IPTG induction significantly influences the proper folding and solubility of recombinant proteins (Huang et al. 2021 ). Lower temperatures (typically 16–25°C) slow down protein synthesis, reducing misfolding and the formation of insoluble inclusion bodies (Chalmers et al. 1990 ; Silaban 2018 ; Mohammadpour-Aghdam et al. 2021 ). Otherwise, induction at higher temperatures (30–37°C) often results in rapid protein synthesis, which can lead to misfolding and loss of function. In contrast (Mühlmann et al. 2017 ). Accordingly, the post-induction temperature was optimized along with the effective IPTG concentration of 0.1 mM, and experimental results 436/540 nm (excitation/emission) of fluorescent emission for 18°C, 30°C, and 37°C are 4.6, 4.3, and 3.8 values, respectively) demonstrated that the most efficient temperature was 18°C (Fig. S8), which is consistent with the literature (Chalmers et al. 1990 ; Silaban 2018 ; Mohammadpour-Aghdam et al. 2021 ). Uncut GFP-Hepc25 complex offers a stable antimicrobial framework Once well-optimization of the GFP-Hepc25, various performance tests were performed to assess its structural integrity and biological activity in various applications. Heat can denature AMPs, leading to the loss of their antimicrobial efficacy by disrupting secondary or tertiary structures (Phambu et al. 2017 ). The stability of the GFP-Hepc25 was examined after exposure to 100°C for 5, 10, and 30-mins intervals. Accordingly, no degradation in stability was observed for GFP-Hepc25 heated for 5 minutes, and 10 minutes at 100°C or those kept at 37°C. However, the peptides exposed to 100°C for 30 minutes showed aggregation, indicating a loss of stability (Fig. 2 ). which is in line with the previously available study performed on the synthetic peptide (Fig. S9)(Dandurand et al. 2014 ). This is supported by the molecular dynamics (MD) simulation that was performed at constant temperatures (40°C and 100°C), suggesting AMPs could lose stability at 100°C having irregular and high fluctuations in RMSD plot during MD simulations at 100°C (Tanhaeian et al. 2020 ). The stability of AMPs under different heavy metal salt treatments is investigated to evaluate their structural and functional stability in environments contaminated with heavy metals. Heavy metals can interact with AMPs, potentially altering their secondary structure, folding, or charge distribution, which may compromise their antimicrobial activity (Ma et al. 2024 ). The stability of the GFP-Hepc25 under different heavy metal salt treatments against E. coli was evaluated, and the MIC values were determined (Fig. 3 A-F). The MIC values for GFP-Hepc25 were measured as 301,07 µg/mL at 37°C and under treatment with MnCl₂, ZnSO₄, MgSO₄, FeSO₄, and CuSO₄. For E. coli , the MIC values were as follows: 285,26 µg/mL, 270,436 µg/mL, 269,11 µg/mL, 399,84 µg/mL, 349,52 µg/mL. These findings indicate that heavy metal salts did not compromise the structural integrity of GFP-Hepc25 or its antimicrobial activity. Furthermore, the relatively higher MIC observed in the presence of FeSO₄ and CuSO₄ may suggest potential interactions between these metals and GFP-Hepc25 due to the capability of binding Cu²+ of hepcidin through the amino-terminal copper–nickel binding motif (Kulprachakarn et al. 2016 ). Overall, heavy metals did not induce degradation of GFP-Hepc25. Likewise, the stability of the GFP-Hepc25 against S. aureus was also evaluated under various heavy metal salt treatments, and the MIC values were determined (Fig. 3 G-L). The results are as follows: (i) the MIC value at 37°C was 379,1 µg/mL. (ii) Under treatment with MnCl₂, the MIC value was 296,99 µg/mL, while ZnSO₄ resulted in a MIC of 227,086 µg/mL, indicating enhanced antimicrobial activity in the presence of these salts, which is an inverse correlation of the report in the literature that Mn exposure causes decreased expressions of hepcidin (Fan et al. 2016 ). Yet, this is in line with the findings that hepcidin attenuates zinc efflux in cells ( Hennigar et al. 2016 ). (iii) MgSO₄ treatments resulted in MIC values of 397,46 µg/mL, suggesting minimal interaction with GFP-Hepc25. Interestingly, (iv) FeSO₄ treatment showed a strong max MIC value of 404,26 µg/mL compared to that of other metals, indicating the effect of Fe on GFP-Hepc25 activity, consistent with studies suggesting that the hepatic peptide hepcidin regulates iron absorption, plasma concentration, and tissue distribution ( Ganz and Nemeth 2013 ). CuSO₄ treatment resulted in a MIC value of 355,64 µg/mL, showing no degradation or loss of activity in the presence of this salt. Collectively, the findings suggest that while GFP-Hepc25 maintains its structural stability and antimicrobial activity under most heavy metal conditions, the presence of ZnSO₄, MnCl₂, and the strong max MIC value of FeSO₄ highlights potential interactions that may enhance its effectiveness. Importantly, no significant degradation of GFP-Hepc25 was observed under any of the tested conditions. Likewise, we also performed stability analysis of GFP-Hepc25 using various chemical treatment to evaluate its structural integrity and functional resilience in acidic environments. The stability of GFP-Hepc25 against E. coli was tested with the following MIC values: 37°C: 297,636 µg/mL; sodium sulfite: 244.086 µg/mL; benzoic acid: 274.142 µg/mL; sodium benzoate: 386.92 µg/mL; sodium chloride: 351,9 µg/mL; acetic acid: 289,986 µg/mL (Fig. 4 A-F). Similarly, the stability of GFP-Hepc25 against S. aureus was evaluated under acid treatment, yielding the following MIC values: 37°C: 387.6 µg/mL; sodium sulfite: 330.004 µg/mL; benzoic acid: 404.94 µg/mL; sodium benzoate: 407.66 µg/mL; sodium chloride: 366,52 µg/mL; acetic acid: 264,452 µg/mL (Fig. 4 G-L). These results show that GFP-Hepc25 remains stable and active under acidic conditions, with enhanced antimicrobial activity in the presence of sodium chloride and acetic acid, well known that the bactericidal activity of Hepc25 increases significantly at acidic pH (Maisetta et al. 2010 ; Lombardi et al. 2015 ). Suggests high antimicrobial activity might be related to the isolated folding yields of human hepcidin under acidic polymer-supported conditions were superior to those under basic folding conditions (Zhang et al. 2010 ). No degradation was observed, which is correlated with the knowledge that hepcidin precipitation is also prevented under acidic conditions (Zhang et al. 2010 ). The stability and antimicrobial activity of the GFP-Hepc25 peptide under detergent treatment were evaluated against E. coli and S. aureus. In the tests performed on E. coli , the stability of GFP-Hepc25 was investigated under different detergent conditions, and MIC values were obtained as follows: 37°C: 265.2 µg/mL, SDS: 24.14 µg/mL, Tween 20: 190 µg/mL, Triton X-100: 174.4 µg/mL (Fig. 5 A-D). Similarly, in the tests conducted on S. aureus , the stability of GFP-Hepc25 under detergent treatment was assessed, and the following MIC values were measured: 37°C: 269 µg/mL, SDS: 510 µg/mL, Tween 20: 285.6 µg/mL, Triton X-100: 74.8 µg/mL (Fig. 5 E-H). This indicates that detergents may disrupt the stability of peptides and inhibit their biological activity. The data were analyzed using linear regression analysis. A p-value of 0.05, which was not statistically significant. We also performed growth inhibition assay to assess the efficacy of GFP-Hepc25 against E. coli (Gram-negative) (Fig. 6 A) and S. aureus (Gram-positive) (Fig. 6 B). The goal was to determine whether GFP-Hepc25 could inhibit bacterial growth in a dose- and time-dependent manner, as well as to compare its activity across different bacterial species. According to the data, the inhibitory effect began at the 5-hour time point, slowing bacterial growth. When compared to the negative control of E. coli , the peptide at a concentration of 500.0 µg/mL caused a significant reduction in growth rate (Fig. 6 ). Similarly, decreasing inhibitory effects were observed at concentrations of 250.0 µg/mL and 125.0 µg/mL compared to 500.0 µg/mL. These results align with the MIC value of GFP-Hepc25 against E. coli (258.264 µg/mL). For S. aureus , comparison with the negative control revealed that the peptide at 500.0 µg/mL also induced a marked decrease in growth rate (Fig. 6 ). Lower concentrations (250.0 µg/mL and 125.0 µg/mL) exhibited reduced inhibition compared to 500.0 µg/mL, consistent with the MIC value for S. aureus (377.74 µg/mL). These findings are crucial for understanding the therapeutic potential of GFP fused Hepc25 and its possible application as a broad-spectrum recombinant antimicrobial complex (Rauf et al. 2020 ; Chachami et al. 2013 ). Data were analyzed using linear regression analysis, with results considered statistically significant at p < 0.05. The SEM analyses revealed that the GFP-Hepc25 peptide exhibited significant antimicrobial effects against E. coli and S. aureus (Fig. 7 ). While no morphological changes were observed in the control group (without peptide) for either bacterial species, deformations in the bacterial membrane were detected in the samples containing GFP-Hepc25. Additionally, it was observed that the bacteria penetrated into one another. These deformations indicate that the peptide binds to the bacterial membrane, causing structural damage. Similar results were obtained for S. aureus . By comparing the bacterial density of peptide-treated and untreated microorganisms, it was observed that the peptide exerts its antimicrobial effect through intercellular activity. In the biofilm inhibition assay, tests conducted against the S. aureus strain showed significant biofilm inhibition at concentrations of 28 and 14 µM, while no inhibitory effect was detected at 9 µM (Fig. 8 A). In contrast, tests performed on the P. aeruginosa demonstrated measurable biofilm inhibition at all concentrations of GFP-Hepc25 (28, 14, and 9 µM) (Fig. 8 B). These results indicate that GFP-Hepc25 has the potential to inhibit biofilm formation in both tested bacterial species, but the efficacy varies depending on the bacterial strain and applied concentration. The data were analyzed using the Student’s t-test, and results with a p-value < 0.05 were considered statistically significant. This study evaluated the cytotoxic effects of GFP-Hepc25 on the L929 fibroblast cell line (Fig. 9). The results demonstrate that GFP-Hepc25 reduces cell viability in a dose- and time-dependent manner. While no significant effect on cell viability was observed at low concentrations (5–20 µM), a marked decrease occurred at 50 µM and higher concentrations. At high doses (150–200 µM), cell death rates reached nearly maximal levels. These findings indicate that GFP-Hepc25 may impair vital cellular functions at certain dose ranges. In this context, the effects of GFP-Hepc25 on cell viability could be evaluated within the framework of iron homeostasis-related biochemical processes. Our study reveals the potential toxic effects of GFP-Hepc25 on the L929 cell line, contributing to a more detailed investigation of this molecule's cellular mechanisms. Conclusion Antibiotic resistance has become a global health issue today. The declining efficacy of traditional antibiotics necessitates the development of next-generation antimicrobial agents. GFP-Hepc25 is a promising candidate in the fight against antibiotic resistance. Its broad-spectrum antimicrobial activity, effectiveness against both Gram-negative and Gram-positive bacteria, and low toxicity make this peptide suitable for pharmaceutical applications. In particular, GFP-Hepc25 can be used in cases such as infections. Additionally, its potential to inhibit biofilm formation could provide a significant advantage in treating chronic infections. In pharmaceutical applications, GFP-Hepc25 may be used in the form of topical creams. The peptide's low toxicity on human cells supports its therapeutic use. GFP-Hepc25 has significant potential for use in various industries, including food, pharmaceuticals, and agriculture, due to the broad-spectrum effects and low toxicity of antimicrobial peptides. This peptide stands out for its effectiveness against both Gram-negative and Gram-positive bacteria, high stability, and low toxicity to human cells. These properties enable GFP-Hepc25 to be widely used in industrial and medical applications. In the food industry, controlling microorganisms that cause food spoilage is of great importance. While chemical preservatives (e.g., benzoic acid, sodium benzoate) are effective, consumers' increasing demand for natural and safe alternatives makes antimicrobial peptides like GFP-Hepc25 an attractive option. GFP-Hepc25 could serve as a natural preservative alternative to traditional chemicals. Its stability in acidic environments and retention of antimicrobial activity support its use as a food preservative. Moreover, the peptide's increased activity in the presence of NaCl makes it even more advantageous for use in salty food products. The use of GFP-Hepc25 in the food industry can enhance food safety while meeting consumer demand for natural products. The agricultural sector is constantly seeking new solutions to control plant diseases and improve crop yields. GFP-Hepc25 could contribute to controlling plant diseases by being used as a fungicide and protective agent in agriculture. Its effectiveness against plant pathogens, in particular, makes this peptide suitable for use in agricultural pesticides. GFP-Hepc25 can be employed to prevent fungal infections in plants and treat existing infections. Additionally, its stability under environmental stress conditions could enhance durability in agricultural applications. The use of GFP-Hepc25 in agriculture may offer a more environmentally friendly alternative to chemical pesticides. The industrial-scale production of GFP-Hepc25 enables its widespread use. High-yield production through culture medium optimization and the use of low IPTG concentrations ensures a cost-effective manufacturing process. Furthermore, incubation at low temperatures (18°C) improves protein folding, increasing soluble protein yield. GFP-Hepc25's stability under various stress conditions, such as high temperatures, heavy metals, and acidic environments, facilitates its use in industrial applications. Its resistance to harsh conditions encountered in food processing and agricultural applications could allow GFP-Hepc25 to be effectively utilized in these fields. The peptide's broad-spectrum antimicrobial activity and low toxicity make it suitable for use in various sectors, including food, pharmaceuticals, and agriculture. In conclusion, this study outlines the process of producing a recombinant peptide using GFP to monitor its expression levels, optimize fermentation conditions, and evaluate its stability and bioactivity. The present work successfully designed, produced, and characterized the recombinant antimicrobial peptide GFP-Hepc25, demonstrating its potential as a novel antimicrobial agent. Declarations Acknowledgements This study was supported by the University of Health Sciences BAP Coordination Office within the scope of the Master's Thesis Project. Author contributions Data availability statement All data supporting the findings of this study are included within the article and its supplementary materials. References Achmüller, C., Kaar, W., Ahrer, K. et al. (2007). Npro fusion technology to produce proteins with authentic N termini in E. coli. Nat Methods , 4, 1037–1043. Albano, C. R., Randers-Eichhorn, L., Bentley, W. E., & Rao, G. (1998). Green fluorescent protein as a real-time quantitative reporter of heterologous protein production. Biotechnol Prog , 14(2), 351-354. Alexander, L., Lee, H., Rosenzweig, M., Jung, J. U., & Desrosiers, R. C. (1997). EGFP-containing vector system that facilitates stable and transient expression assays. Biotechniques , 23(1), 64-66. Alfasi, S., Sevastsyanovich, Y., Zaffaroni, L., Griffiths, L., Hall, R., & Cole, J. (2011). Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins. J Biotechnol , 156(1), 11-21. Arezes, J., & Nemeth, E. (2015). Hepcidin and iron disorders: new biology and clinical approaches. Int J Lab Hematol , 37(S1), 92-98. Cha, H. J., Dalal, N. G., Pham, M. Q., Vakharia, V. N., Rao, G., & Bentley, W. E. (1999). Insect larval expression process is optimized by generating fusions with green fluorescent protein. Biotechnol Bioeng , 65(3), 316-324. Cha, H. J., Dalal, N. G., Vakharia, V. N., & Bentley, W. E. (1999). Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells. J Biotechnol , 69(1), 9-17. Cha, H. J., Pham, M. Q., Rao, G., & Bentley, W. E. (1997). Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. Biotechnol Bioeng , 56(3), 239-247. Cha, H. J., Wu, C. F., Valdes, J. J., Rao, G., & Bentley, W. E. (2000). Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility. Biotechnol Bioeng , 67(5), 565-574. Chachami, G., Lyberopoulou, A., Kalousi, A., Paraskeva, E., Pantopoulos, K., & Simos, G. (2013). Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera. Biochem Biophys Res Commun , 435(4), 540-545. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science , 263(5148), 802-805. Chalmers, J. J., Kim, E., Telford, J. N., Wong, E. Y., Tacon, W. C., Shuler, M. L., & Wilson, D. B. (1990). Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor. Appl Environ Microbiol , 56(1), 104-111. Choyam, S., Jain, P. M., & Kammara, R. (2021). Characterization of a potent new-generation antimicrobial peptide of Bacillus. Front Microbiol , 12, 710741. Dandurand, J., Samouillan, V., Lacoste-Ferré, M. H., Lacabanne, C., Bochicchio, B., & Pepe, A. (2014). Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: Signature of the amyloid fibers. Pathol Biol , 62(2), 100-107. Durdagi, S., Dağ, Ç., Dogan, B., Yigin, M., Avsar, T., Buyukdag, C., ... & DeMirci, H. (2021). Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing. Structure , 29(12), 1382-1396. Fahimirad, S., Ghaznavi-Rad, E., Abtahi, H. et al. (2021). Antimicrobial Activity, Stability and Wound Healing Performances of Chitosan Nanoparticles Loaded Recombinant LL37 Antimicrobial Peptide. Int J Pept Res Ther , 27, 2505–2515. Fan, Q., Zhou, Y., Yu, C., Chen, J., Shi, X., Zhang, Y., & Zheng, W. (2016). Cross-sectional study of expression of divalent metal transporter-1, transferrin, and hepcidin in blood of smelters who are occupationally exposed to manganese. PeerJ , 4, e2413. Gagliardo, B., Faye, A., Jaouen, M., Deschemin, J. C., Canonne-Hergaux, F., Vaulont, S., & Sari, M. A. (2008). Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone. FEBS J , 275(15), 3793-3803. Ganz, T. (2011). Hepcidin and iron regulation, 10 years later. Blood , 117(17), 4425-4433. Ganz, T., & Nemeth, E. (2012). Hepcidin and iron homeostasis. Biochim Biophys Acta , 1823(9), 1434-1443. Hennigar, S. R., & McClung, J. P. (2016). Hepcidin attenuates zinc efflux in Caco-2 cells. J Nutr , 146(11), 2167-2173. Huang, C. J., Peng, H. L., Patel, A. K., Singhania, R. R., Dong, C. D., & Cheng, C. Y. (2021). Effects of lower temperature on expression and biochemical characteristics of HCV NS3 antigen recombinant protein. Catalysts , 11(11), 1297. Kulprachakarn, K., Chen, Y. L., Kong, X. et al. (2016). Copper(II) binding properties of hepcidin. J Biol Inorg Chem , 21, 329–338. Krause, A., Neitz, S., Magert, H. J., et al. (2000). LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett , 480(2-3), 147-150. Larentis, A. L., Nicolau, J. F. M. Q., Esteves, G. d. S. et al. (2014). Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Res Notes , 7, 671. Lecina, M., Sarró, E., Casablancas, A., Gòdia, F., & Cairó, J. J. (2013). IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions. Biochem Eng J , 70, 78-83. Lombardi, L., Maisetta, G., Batoni, G., & Tavanti, A. (2015). Insights into the antimicrobial properties of hepcidins: advantages and drawbacks as potential therapeutic agents. Molecules , 20(4), 6319-6341. Luo, X., Song, Y., Cao, Z., Qin, Z., Dessie, W., He, N., ... & Tan, Y. (2022). Evaluation of the antimicrobial activities and mechanisms of synthetic antimicrobial peptide against food-borne pathogens. Food Biosci , 49, 101903. Ma, X., Wang, Q., Ren, K., Xu, T., Zhang, Z., Xu, M., Rao, Z., & Zhang, X. (2024). A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies. Fermentation , 10(11), 540. Maisetta, G., Di Luca, M., Esin, S., Florio, W., Brancatisano, F. L., Bottai, D., Campa, M., & Batoni, G. (2008). Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3. Peptides , 29(1), 1-6. Maisetta, G., Petruzzelli, R., Brancatisano, F. L., Esin, S., Vitali, A., Campa, M., & Batoni, G. (2010). Antimicrobial activity of human hepcidin 20 and 25 against clinically relevant bacterial strains: effect of copper and acidic pH. Peptides , 31(11), 1995-2002. Maisetta, G., Vitali, A., Scorciapino, M. A., Rinaldi, A. C., Petruzzelli, R., Brancatisano, F. L., ... & Batoni, G. (2013). pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25. FEBS J , 280(12), 2842-2854. Mohammadpour-Aghdam, M., Molaeirad, A., & Azizi, A. (2021). The Effect of DMSO, IPTG and Incubation Temperature on Growth Hormone Protein Expression in E. coli. Biomacromol J , 7(3), 86-92. Mühlmann, M., Forsten, E., Noack, S. et al. (2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Fact , 16, 220. Olaofe, O. A., Burton, S. G., Cowan, D. A., & Harrison, S. T. (2010). Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli. Biochem Eng J , 52(1), 19-24. O’Toole, G. A. (2011). Microtiter dish biofilm formation assay. J Vis Exp , 47, e2437. Phambu, N., Almarwani, B., Garcia, A. M., Hamza, N. S., Muhsen, A., Baidoo, J. E., & Sunda-Meya, A. (2017). Chain length effect on the structure and stability of antimicrobial peptides of the (RW)n series. Biophys Chem , 227, 8-13. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., & Cormier, M. J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene , 111(2), 229-233. Rauf, A., Shariati, M. A., Khalil, A. A., Bawazeer, S., Heydari, M., Plygun, S., ... & Aljohani, A. S. (2020). Hepcidin, an overview of biochemical and clinical properties. Steroids , 160, 108661. Riss, T. L., et al. (2013). Cell Viability Assays. Eli Lilly & Company and the National Center for Advancing Translational Sciences. Rochette, R., Gudjoncik, A., Guenancia, C., Zeller, M., Cottin, Y., & Vergely, C. (2015). The iron-regulator hormone hepcidin: a possible therapeutic target? Pharmacol Ther , 146, 35-52. Rücker, E., Schneider, G., Steinhäuser, K., Löwer, R., Hauber, J., & Stauber, R. H. (2001). Rapid evaluation and optimization of recombinant protein production using GFP tagging. Protein Expr Purif , 21(1), 220-223. Sadr, V., Saffar, B., & Emamzadeh, R. (2017). Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology. Gene , 610, 112-117. Shike, H., Lauth, X., Westerman, M. E., Ostland, V. E., Carlberg, J. M., Van Olst, J. C., Shimizu, C., Bulet, P., & Burns, J. C. (2002). Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. Eur J Biochem , 269(8), 2232-2237. Shimomura, O., Johnson, F. H., & Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol , 59(3), 223-239. Silaban et al. (2018). IOP Conf. Ser.: Earth Environ. Sci., 217, 012039. Tanhaeian, A., Mohammadi, E., Mansury, D., & Zeinali, T. (2020). Assessment of a novel antimicrobial peptide against clinically isolated animal pathogens and prediction of its thermal-stability. Microb Drug Resist , 26(4), 412-419. Venkatesh, B., Arifuzzaman, M., Mori, H., Suzuki, S., Taguchi, T., & Ohmiya, Y. (2005). Use of GFP tags to monitor localization of different luciferases in E. coli. Photochem Photobiol Sci , 4(9), 740-743. Waldo, G., Standish, B., Berendzen, J. et al. (1999). Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol , 17, 691–695. Wiegand, I., Hilpert, K., & Hancock, R. E. W. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc , 3(2), 163-175. Winterbourn, C. C. (1995). Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett , 82-83, 969-974. Zhang, H., Yuan, Q., Zhu, Y., & Ma, R. (2005). Expression and preparation of recombinant hepcidin in Escherichia coli. Protein Expr Purif , 41(2), 409-416. Zhang, J., Diamond, S., Arvedson, T., Sasu, B. J., & Miranda, L. P. (2010). Oxidative folding of hepcidin at acidic pH. Biopolymers , 94(2), 257-264. Zhao, F., Yang, N., Wang, X., Mao, R., Hao, Y., Li, Z., ... & Wang, J. (2019). In vitro/vivo mechanism of action of MP1102 with low/nonresistance against Streptococcus suis type 2 strain CVCC 3928. Front Cell Infect Microbiol , 9, 48. Zuo, X., Mattern, M. R., Tan, R., Li, S., Hall, J., Sterner, D. E., Shoo, J., Tran, H., Lim, P., Sarafianos, S. G., Kazi, L., Navas-Martin, S., Weiss, S. R., & Butt, T. R. (2005). Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif , 42(1), 100-110. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6555765","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452227086,"identity":"0c968a9a-e1b4-4e39-b557-2e2044ba418e","order_by":0,"name":"Gamze Balcı","email":"","orcid":"","institution":"University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gamze","middleName":"","lastName":"Balcı","suffix":""},{"id":452227087,"identity":"4ca97865-ffd9-41ea-8c7f-d37df2a77253","order_by":1,"name":"Esra Ayan","email":"","orcid":"","institution":"University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Ayan","suffix":""},{"id":452227088,"identity":"68606496-df07-41dc-b5c7-464eae15ed5c","order_by":2,"name":"Ahmet Katı","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCTCZwMDAzHyAGcw+QIyWA0AtPMxsCTAtjA3EaWHgMSBOC//s7sTHHxjS5O3ZeT5/LmxjkOO7kcD+uAKfJXfObjY4wJBj2MPMu016ZhuDseSNBMbGM/isuZG7TeIAQwUjSAszbxtD4gaQFnwuk7+Ru/0HUIt9DzPP489ALfUEtRgAbQF6OCcRqIVBGqglwYCQFsMbuZslzhikJfccZjOTnnFOwnDmmYeNM/FpkbuRu/FDRUWybXv/4cefC8ps5PmOJx/4iE8L1HlwFiiaCMTkKBgFo2AUjALCAABU408rc+jKSwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Katı","suffix":""}],"badges":[],"createdAt":"2025-04-29 11:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6555765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6555765/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82364136,"identity":"bb54ac37-c51b-4a7c-91fd-7b0adbb4f7b6","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":276889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative antimicrobial activity performance between GFP-Hepc25 and Hepc25 peptides. (A,B) \u003c/strong\u003eMinimum inhibitor concentration trends of GFP-Hepc25 and Hepc25 alone, in\u003cem\u003e E. coli\u003c/em\u003e trial.\u003cstrong\u003e (C,D)\u003c/strong\u003e Minimum inhibitor concentration trends of GFP-Hepc25 and Hepc25 alone, in\u003cem\u003eS. aureus\u003c/em\u003e trial. There is no statistically significant difference in antimicrobial activity between GFP-Hepc25 and Hepc25 alone against \u003cem\u003eE. coli\u003c/em\u003e, but, there is in \u003cem\u003eS. aureus\u003c/em\u003e. PC: positive control, NC: negative control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/2573e1be3551a04535914105.png"},{"id":82364137,"identity":"051f3372-3d00-4dd3-a391-93a38349759c","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":406240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThermal stability test of GFP-Hepc25 against E. coli and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e S. aureus\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Antimicrobial activity of GFP-Hepc25 after incubation at different temperatures against \u003cem\u003eE. coli\u003c/em\u003e. MIC assay shows that the lower peptide concentration in the 100°C-5-min test is correlated with weak antimicrobial activity. No minimal antimicrobial activity was observed in other tests even.\u003cstrong\u003e (B) \u003c/strong\u003eAntimicrobial activity of GFP-Hepc25 after incubation at different temperatures against \u003cem\u003eS. aureus\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e MIC assay showed similar consequences to panel A, suggesting peptides exposed to 100°C for 10 and 30 minutes showed a loss of antimicrobial activity, indicating a loss of stability.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/5541b2c51eab27bfaf36d956.png"},{"id":82364708,"identity":"63285551-d96d-4bf3-a763-9c40052514bd","added_by":"auto","created_at":"2025-05-09 12:38:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":840385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative result of heavy metal stability test of GFP-Hepc25. (A-F) \u003c/strong\u003eAntimicrobial activities of GFP-Hepc25 treated with six different metals against \u003cem\u003eE. coli\u003c/em\u003e and the control group where the GFP-Hepc25 were not treated with heavy metals (panel F).\u003cstrong\u003e (G-L)\u003c/strong\u003e Antimicrobial activities of GFP-Hepc25 treated with six different metals against \u003cem\u003eS. aureus\u003c/em\u003e and the control group where the GFP-Hepc25 were not treated with heavy metals (panel L).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/bbb4f8643b90ea91b929f9d8.png"},{"id":82364141,"identity":"84be80b5-9478-41dc-9f6d-c2aa074a9d04","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":865802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative result of chemical environment stability test of GFP-Hepc25. (A-F)\u003c/strong\u003e Antimicrobial activities of GFP-Hepc25 treated with five different chemicals against \u003cem\u003eE. coli\u003c/em\u003e, and the control group where the GFP-Hepc25 were not treated with any chemicals (panel F). \u003cstrong\u003e(G-L) \u003c/strong\u003eAntimicrobial activities of GFP-Hepc25 treated with five different chemicals against \u003cem\u003eS. aureus\u003c/em\u003e, and the control group where the GFP-Hepc25 were not treated with any chemical (panel L).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/d67a1efd666ac4e76d27425b.png"},{"id":82364147,"identity":"ca6d57bd-5d5a-416a-8744-d2dca0226410","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative result of alkaline environment stability test of GFP-Hepc25. \u003c/strong\u003eAntimicrobial activities of GFP-Hepc25 treated with three different detergents against\u003cem\u003e E. coli\u003c/em\u003eand \u003cem\u003eS. aureus\u003c/em\u003e, and the control group where the GFP-Hepc25 were not treated with detergents (panels D and H).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/a13aedeb5d96599ba69dd952.png"},{"id":82364142,"identity":"31b498ff-9991-40a4-b0c1-b85135717410","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":182994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth Inhibition Assay graph against (A)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e E. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and (B) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eIncreasing concentrations of GFP-Hepc25 effectively inhibit bacterial growth, with significant reductions in absorbance at both T20, T22, and T24 time points compared to the initial measurement (T0). This trend is more pronounced at higher AMP concentrations, indicating a dose-dependent inhibition effect. The data confirm that GFP-Hepc25 exhibits strong antimicrobial activity against both Gram-negative \u003cstrong\u003e(A)\u003c/strong\u003e and Gram-positive\u003cstrong\u003e (B) \u003c/strong\u003ebacteria, maintaining efficacy over time. Positive (PC) and negative (NC) controls validate the specificity of the observed inhibition.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/1cdc9dfda4d90e42b9e4f9cb.png"},{"id":82364151,"identity":"e3ed4d9c-3383-4134-8720-802011010f1d","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":656835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of the GFP-Hepc25 peptide against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eThe magnifications from left to right are as follows: 30,000x, 50,000x, 100,000x, and 200,000x. (\u003cstrong\u003eA,B,C,D)\u003c/strong\u003ebelong to the\u003cem\u003e E. coli \u003c/em\u003econtrol group. (\u003cstrong\u003eE,F,G,H)\u003c/strong\u003e belong to the \u003cem\u003eE. coli \u003c/em\u003egroup treated with GFP-Hepc25.\u003cstrong\u003e (I,J,K,L)\u003c/strong\u003e belong to the\u003cem\u003e S. aureus\u003c/em\u003e control group.\u003cstrong\u003e (M,N,O,P)\u003c/strong\u003e belong to the\u003cem\u003e S. aureus\u003c/em\u003e group treated with GFP-Hepc25.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/ab61fcfb51d138896d5704ce.png"},{"id":82364143,"identity":"cfa61995-185a-49c3-9f0e-a8100bedb8a3","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":131038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiofilm Inhibition Assay Graphs. (A)\u003c/strong\u003e Biofilm inhibition graph of GFP-Hepc25 against \u003cem\u003eS. aureus. \u003c/em\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Biofilm inhibition graph of GFP-Hepc25 against\u003cem\u003e P. aeruginosa.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/acc84918fc513147a68a87d5.png"},{"id":82364139,"identity":"b3ba44db-8ab3-499c-8f34-56ca05bb7766","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":94065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the Cytotoxicity Test of GFP-Hepc25 on the L929 Cell Line.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/82d51a0a87bbb41b1ff378ef.png"},{"id":86884849,"identity":"bf7729ed-b7f1-407c-85aa-4596ce4ce5e3","added_by":"auto","created_at":"2025-07-16 17:31:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4974278,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/c0648336-192f-4b93-9a50-c7affa4e8764.pdf"},{"id":82364159,"identity":"56f7db00-8463-4ca9-94cb-8acc80fd4fe5","added_by":"auto","created_at":"2025-05-09 12:30:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5827498,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/139b012cea11656cc9289f97.docx"},{"id":82365749,"identity":"5891a69f-da3a-47af-8d8d-b33bb17ccbc3","added_by":"auto","created_at":"2025-05-09 12:46:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":714711,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555765/v1/93366b5e5dc12225763be424.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepcidin was discovered as the liver-expressed antimicrobial peptide in 2000 (Krause et al. 2000). Hepcidin is encoded by the hepcidin antimicrobial peptide (\u003cem\u003eHAMP\u003c/em\u003e) gene. It is first produced as an 84-amino acid pre-pro-hepcidin, which is then processed into a 60-amino acid pro-hepcidin. Finally, it is sliced to a mature C-terminal 25 amino acid active peptide (Rochette et al. 2015). Hepcidin is a crucial regulator of iron homeostasis in the body, mainly by controlling the expression of ferroportin-1 (FPN-1), an iron-exporting protein found on duodenal enterocytes and macrophages (Ganz and Nemeth 2012). Hepcidin-25 (Hepc25), one of its major and active isoforms, plays a critical role in both iron metabolism and innate immunity. Structurally, it consists of 25 amino acids and possesses a distinctive \u0026beta;-hairpin loop stabilized by four disulfide bonds, which contributes to its biological activity (Rauf et al. 2020). The principal function of Hepc25 is to regulate iron levels by binding to the iron exporter ferroportin, found on the surface of enterocytes, macrophages, and hepatocytes. Once bound, Hepc25 induces the internalization and degradation of ferroportin, reducing the release of iron into circulation. This process is crucial in preventing excessive iron accumulation, which could otherwise catalyze the formation of harmful free radicals through the Fenton reaction (Winterbourn 1995). Hepc25 expression is modulated by factors such as inflammation, hypoxia, and iron levels. During infection or inflammation, cytokines like IL-6 upregulate Hepc25 expression, leading to iron sequestration (a mechanism known as the anemia of inflammation). Consequently, dysregulation of Hepc25 can result in iron-related disorders such as anemia of chronic disease or iron overload conditions like hereditary hemochromatosis (Arezes and Nemeth 2015). Beyond its role in iron regulation, Hepc25 exhibits antimicrobial properties. By limiting iron availability, it creates a hostile environment for pathogens that require iron for survival and growth. Additionally, Hepc25 has recently gained attention as a potential antimicrobial agent with therapeutic applications for treating various infectious diseases; it is directly bactericidal activity against several microorganisms, reinforcing its role in the innate immune response (Ganz 2011). \u003c/p\u003e\n\u003cp\u003eHuman Hepc25 was successfully expressed recombinantly in \u003cem\u003eEscherichia coli\u003c/em\u003e. However, since the bacterial cytosol cannot form and maintain disulfide bonds, an additional in vitro refolding step was necessary to fold the peptide properly (Zhang et al. 2005; Achm\u0026uuml;ller et al. 2007). To address this challenge, the \u003cem\u003eE. coli\u003c/em\u003e Origami strain was employed (Gagliardo et al. 2008). The bacterial expression system is straightforward, requires minimal time for DNA manipulation, and typically achieves high protein expression levels. While the \u003cem\u003eE. coli\u003c/em\u003e expression system offers several advantages, it also comes with notable limitations. These include challenges like codon usage bias, proteolytic degradation of the expressed protein, difficulties in protein release, and potential toxicity of the expressed peptides to the host cells. Notably, non-fusion Hepc25 is often produced as inclusion bodies, necessitating complex and time-intensive refolding steps to recover appropriately folded protein structures (Zhang et al., 2005). Various protein fusion technologies have been developed to enhance the expression of target peptides in host cells, including glutathione-S-transferase, small ubiquitin-related modifier protein, and thioredoxin gene fusion systems (Sadr et al. 2017). Despite their utility, these systems may still face challenges, such as inconsistent expression levels and reduced efficiency (Zuo et al. 2005). Otherwise, green fluorescent protein (GFP) appears as a unique protein marker due to its two key advantages that \u003cstrong\u003e(i)\u003c/strong\u003e fluorescence of GFP without the need for additional co-factors or staining, and \u003cstrong\u003e(ii)\u003c/strong\u003e fluorescence of GFP is easily observable from outside the cells (Chalfie et al. 1994). Initially discovered in the jellyfish \u003cem\u003eAequorea victoria\u003c/em\u003e, GFP was first identified and described in 1962 (Shimomura et al. 1962), and its cDNA was successfully cloned in 1992 (Prasher et al. 1992). Recently, GFP fusion technology has been applied to enhance protein production in \u003cem\u003eE. coli\u003c/em\u003e. Fusing GFP to the N-terminus of target proteins improves protein solubility, expression levels, and purification efficiency, making valuable tool of GFP use for recombinant protein expression (Cha et al. 2000). Earlier studies have highlighted using GFP as a quantitative marker for protein expression in insect cell systems (Cha et al. 1999) and insect larvae (Cha et al. 1997, Cha et al. 1999). Additionally, research demonstrated its utility in \u003cem\u003eE. coli\u003c/em\u003e fermentation processes, where GFP and the target protein, chloramphenicol acetyltransferase (CAT), were co-expressed in an operon system with identical ribosome binding sites for both genes (\u003cem\u003eGFP\u003c/em\u003e and \u003cem\u003ecat\u003c/em\u003e) (Albano et al. 1998). A study conducted in 1999 further validated GFP as a noninvasive, quantitative fusion marker for monitoring foreign protein production in recombinant \u003cem\u003eE. coli\u003c/em\u003e (Cha et al. 2000). In a study, a GFP-hepcidin chimera was created by fusing GFP to the C-terminus of hepcidin, and the resulting construct was expressed in Huh7 eukaryotic cells (Chachami et al. 2013). Stable, low-level expression facilitated the secretion of correctly matured hepcidin-GFP. The secreted chimera was demonstrated to be active in regulating iron homeostasis. Under hypoxic conditions, the cellular localization of pro-hepcidin-GFP was altered, and the secretion of mature hepcidin-GFP was significantly reduced. To the best of our knowledge, the potential of GFP fusion technology for producing recombinant human Hepc25 has not been thoroughly explored. Thus, this study aimed to establish a robust method for producing soluble, functional recombinant human Hepc25 by employing codon optimization and a novel GFP-based strategy in an \u003cem\u003eE. coli\u003c/em\u003e expression system. Following purification, the peptide\u0026apos;s characteristics were evaluated using various assays to confirm its functionality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eMaterials, and Bacteria Strains\u003c/h2\u003e \u003cp\u003eThe strains \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3), \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC25923, \u003cem\u003eP. aeruginosa\u003c/em\u003e ATCC9027 were obtained from the University of Health Sciences. Bacteria were cultured in LB (Luria-Bertani broth Lennox, BD Difco, Canada) at 37◦C. Pierce\u0026trade; BCA Protein Assay Kit, and HisPur\u0026trade; Ni-NTA Resin was purchased from Thermo Scientific (Thermo Scientific\u0026trade;, USA). The designed plasmid is provided by Twist Bioscience (twistbioscience.com, South San Francisco).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the fusion expression plasmid pET28a(+)-GFP-Hepc25\u003c/h2\u003e \u003cp\u003eThe antimicrobial peptide was designed using SnapGene, followed by recombinant production. The genes encoding the protein were cloned into the pET28(a) vector with a 6-histidine tag fusion through a service provided by Twist Bioscience (twistbioscience.com). The plasmid's gene sequence consists of the 6-histidine tag, green fluorescent protein, TEV fusion protein, and the Hepc25 sequence (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). The plasmid was then transformed into the \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) for expression testing. Based on the peptide sequence, GFP-Hepc25 was determined to be approximately 34 kDa, while Hepc25 was 6 kDa.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHepcidin25 Amino Acid Sequence\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepcidin25 Amino Acid Sequence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDTHFPICIFCCGCCHRSKCGMCCKT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of Test Expression\u003c/h3\u003e\n\u003cp\u003eThe GFP-Hepc25, ordered in a lyophilized form, was reconstituted by adding 1,000 \u0026micro;l of RNase and DNase-free water. The GFP-Hepc25 was then successfully transformed into a competent \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) following the Durdagi et al. study (Durdagi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Briefly, the GFP-Hepc25 and \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) mixture was incubated on ice for 20 minutes. In a sterile Eppendorf tube, 15 \u0026micro;l of bacterial cells and 3 \u0026micro;l of plasmid DNA were combined, followed by an additional incubation on ice for 30 minutes. To facilitate the transformation, the mixture underwent a heat shock at 42\u0026deg;C for 30 seconds, immediately followed by cooling on ice for 5 minutes. Subsequently, 400 \u0026micro;l of LB broth medium (g/L Tryptone 10.0, Yeast Extract 5.0, Sodium Chloride 5.0) was added to the tube, and the suspension was incubated at 37\u0026deg;C for 1 hour. The bacterial culture was then plated onto LB agar (g/L Tryptone 10.0, Yeast Extract 5.0, Sodium Chloride 5.0), Agar 10.0) containing kanamycin (50 ug/mL) and incubated at 37\u0026deg;C for 16 to 24 hours. The next day, the transformed colonies were cultured in an LB broth medium with kanamycin (50 ug/mL) to ensure selection. Glycerol stocks of the successfully transformed colonies were prepared for long-term storage.\u003c/p\u003e\n\u003ch3\u003eOptimization of Growth Media by Micro-scale Fermentation\u003c/h3\u003e\n\u003cp\u003eIn order to determine the optimal production conditions during fermentation, 11 different media were used (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The optimization conducted using the BioLector microbioreactor (Beckman Coulter, BiolectorPro, USA) involved the following media and their compositions:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe carbon, nitrogen, and mineral source components of the medium used for optimization\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eCarbon Source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eNitrogen Source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eMinerals\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients (g/L) /Number\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeptone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTryptone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDekstrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGelatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYeast Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBeef Extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePapain digest of soybean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNaCl \u0026amp; Dipotassium Phosphate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3,0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5,0 \u0026amp; 2,5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese media were selected to examine the effects of carbon and nitrogen sources, as well as the impact of NaCl concentration (ionic environment) on GFP-Hepc25 production. The first eight media were prepared to compare the effect of different nitrogen sources (casein, peptone, glucose, tryptone) with yeast extract as a constant carbon source. The effect of varying NaCl concentrations was also evaluated. Additionally, formulations containing beef extract, Papain digest of soybean, and dipotassium phosphate were incorporated into the study to evaluate their effects on microbial growth and GFP-Hepc25 production. In this study, medium No. 7 (LB - Luria-Bertani broth), medium No. 10 (NB - Nutrient broth), and medium No. 11 (TSB - Tryptic soy broth) were commercially procured.\u003c/p\u003e \u003cp\u003eThe Rosetta strain was inoculated into kanamycin-containing media. The culture was incubated in a shaking incubator at 37\u0026deg;C and 150 rpm for 1\u0026ndash;2 hours. When the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e nm) reached approximately 0.4, induction was performed with 0.4 mM IPTG. Subsequently, the cultures were incubated in a bioreactor at 18\u0026deg;C with agitation at 800 rpm for 24 hours. Following incubation, the collected samples were centrifuged at 4,000 \u0026times; g for 30 minutes. The pellet was then resuspended in 200 \u0026micro;l of Phosphate-Buffered Saline (PBS). The collected samples were analyzed by electrophoresis on a 15% SDS-PAGE gel.\u003c/p\u003e\n\u003ch3\u003eOptimization of IPTG Concentration with Incubation Time\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) was inoculated into a medium-II (optimized media, scenario 2, containing Casein 10 g/L, Yeast Extract 10 g/L, NaCl 10 g/L) with kanamycin. The culture was incubated in a shaking incubator at 37\u0026deg;C and 150 rpm for 16\u0026ndash;24 hours. In the fermentation cultures prepared in the microbioreactor, the final IPTG concentrations were adjusted to 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 1.0 mM. The cultures were then incubated at 18\u0026deg;C and 800 rpm for 20 hours. The protein production was monitored over a 20-hour period. The positive control consisted of a bacteria-free medium-II, while the negative control was a bacterial strain not induced with IPTG.\u003c/p\u003e\n\u003ch3\u003eOptimization of Incubation Temperature After Induction\u003c/h3\u003e\n\u003cp\u003eTo optimize GFP-Hepc25 production, fermentation cultures (100 mL each, prepared in Erlenmeyer flasks) were incubated at different temperatures to assess the impact of temperature on production yield. Cultures reaching an OD\u003csub\u003e600\u003c/sub\u003e nm value of approximately 0.4 were induced with 0.1 mM IPTG and then incubated at 18\u0026deg;C, 30\u0026deg;C, and 37\u0026deg;C in a shaking incubator for 24 hours. After incubation, samples were collected and centrifuged at 4000 xg for 30 minutes. The fluorescence emission intensity of the samples was measured using a Multimode Reader (Agilent Technologies, BioTek Synergy Neo2, USA). The fluorescence emission intensities were compared, utilizing the green fluorescent protein, to determine the optimal conditions for production. Samples were taken before and after centrifugation, as well as from the supernatant and pellet fractions, followed by 15% SDS. The thickness of SDS-PAGE bands was visually distinguishable, allowing for a clear comparison of protein expression levels between samples, leading to the selection of the optimum incubation temperature.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLarge-scale Fermentation Culture\u003c/h2\u003e \u003cp\u003eBacteria that include GFP-Hepc25 plasmid were inoculated into 300 mL of media-II supplemented with kanamycin (50 ug/mL). After incubation at 37\u0026deg;C with shaking at 150 rpm for 16\u0026ndash;24 hours, 250 mL of this pre-culture was transferred into 5 L of bioreactor (500 rpm, 37\u0026deg;C) medium with kanamycin (50 ug/mL). Once the OD\u003csub\u003e600\u003c/sub\u003e nm reached 0.4, the culture was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The fermentation was carried out in a shaking incubator at 18\u0026deg;C and 250 rpm for 16\u0026ndash;24 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein Purification\u003c/h3\u003e\n\u003cp\u003eFollowing fermentation, the peptides present in the culture were purified. The samples were first centrifuged at 4,000 \u0026times; g for 30 minutes. After discarding the supernatant, the pellet was prepared for subsequent processing steps. A lysis buffer was prepared containing 500 mM NaCl, 50 mM Tris-HCl, 20 mM imidazole, 5% glycerol, and 0.4% Triton X-100. This buffer was stored at +\u0026thinsp;4\u0026deg;C, with 5 mM β-mercaptoethanol (BME) added immediately prior to use. The pellets were resuspended in 45 mL of lysis buffer and subjected to three rounds of sonication for 30 seconds each at 70% power while maintained on ice. The lysate was then centrifuged at 10,000 rpm for 30 minutes. The resulting supernatant was purified using nickel-NTA affinity chromatography via Fast Protein Liquid Chromatography (FPLC). The purification process involved sequential application of equilibration buffer (25 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0), sample loading, and wash buffer (25 mM Tris-HCl, 150 mM NaCl, 40 mM imidazole, pH 8.0), followed by elution with elution buffer (25 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0) into sterile collection tubes. The column was subsequently regenerated using 0.5 M NaOH followed by 20% ethanol and stored at +\u0026thinsp;4\u0026deg;C. For downstream applications, the isolated peptides were dialyzed against dialysis buffer (25 mM Tris-HCl, 150 mM NaCl) using a 10 kDa molecular weight cut-off membrane to remove imidazole, with overnight incubation at +\u0026thinsp;4\u0026deg;C under constant stirring. Finally, the purified peptides were supplemented with 10% glycerol, aliquoted, and stored at -80\u0026deg;C for long-term preservation.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial Activity Assay of GFP-Hepc25 and Hepc25\u003c/h3\u003e\n\u003cp\u003eThe antimicrobial activity of GFP-Hepc25 and Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was determined using the Minimum Inhibitory Concentration (MIC) assay (Wiegand et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The concentration of GFP-Hepc25 and Hepc25 were calculated using the BCA Protein Assay kit. In the MIC test, 50 \u0026micro;L of Mueller Hinton Broth medium (MHB) (containing per liter: 2.0 g beef infusion solids, 17.5 g casein hydrolysate, and 1.5 g starch) was added to each well of a 96-well plate. An initial 100 \u0026micro;L aliquot of the antimicrobial peptide (AMP) was added to the first column and subjected to two-fold serial dilution up to the 10th column. Subsequently, 50 \u0026micro;L of microbial suspension, standardized to 0.5 McFarland (1\u0026times;10⁸ CFU/mL), was inoculated into each well. Following 24-hour incubation at 37\u0026deg;C, the OD\u003csub\u003e600\u003c/sub\u003e nm was measured using a spectrophotometer. The negative control consisted of microorganisms without GFP-Hepc25 or Hepc25, while the positive control contained only MHB medium. This experimental setup allowed for accurate determination of the minimum peptide concentrations required to inhibit visible growth of the test microorganisms.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStability Analysis of GFP-Hepc25 in Selective Heat Treatment\u003c/h2\u003e \u003cp\u003eThe stability of the peptide was examined after exposure to 100\u0026deg;C for different time intervals. In an Eppendorf tube, 1 mL of the GFP-Hepc25 (800,0 \u0026micro;g/mL) was subjected to 100\u0026deg;C for 5, 10, and 30 minutes using a heat block (Fahimirad et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a control, the peptide was kept at 37\u0026deg;C. After heating, the peptides were placed on ice and subjected to MIC testing against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The MIC test is performed by preparing serial dilutions of the GFP-Hepc25 in a growth medium, inoculating each with the test microorganism, and incubating under suitable conditions. The MIC is the lowest concentration of GFP-Hepc25 that prevents visible microbial growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStability Analysis of GFP-Hepc25 in Selective Heavy Metal Treatment\u003c/h2\u003e \u003cp\u003eThe stability of the peptide against various metal salts was assessed by incubating the peptide with the salts at 37\u0026deg;C for 1 hour. For this purpose, MgSO₄, FeSO₄, MnCl₂, ZnSO₄, and CuSO₄ were used (Choyam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with each metal salt adjusted to a final concentration of 1.0 mg/mL. The peptides were mixed with the metal salts in Eppendorf tubes and maintained at a constant temperature for the specified duration. As a control, the peptide was incubated under the same conditions without exposure to the metal salts. After incubation, the antimicrobial activity of the peptides was evaluated using the MIC test on \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e as indicator microorganisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStability Analysis of GFP-Hepc25 in Selective Chemical Treatment\u003c/h2\u003e \u003cp\u003eSolutions of sodium chloride, acetic acid, ascorbic acid, benzoic acid, sodium benzoate, and sodium sulfite were prepared at different concentrations (Choyam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Each (1 mg) preservative was mixed with the (1 mg/mL) GFP-Hepc25 and incubated at 37\u0026deg;C for a minimum of 2 hours. As a control group, the GFP-Hepc25 was incubated under the same conditions without exposure to any food preservatives. After incubation, the antimicrobial activities of the peptides were measured using the MIC test on indicator microorganisms such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStability Analysis of GFP-Hepc25 in Selective Detergent Treatment\u003c/h2\u003e \u003cp\u003eThe stability of the peptide against various detergents and solvents was evaluated by incubating it at 37\u0026deg;C for 5 hours (Choyam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For this purpose, solutions containing 1% sodium dodecyl sulfate (SDS), Tween 80, and Triton X-100 were prepared. Each solution was mixed with a 1 mg/mL peptide sample and incubated under the appropriate conditions. As a control group, the peptide was incubated at the same temperature without exposure to any detergents or solvents. After the incubation period, the antimicrobial activity of the peptides was assessed using the MIC test on indicator microorganisms such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStability Analysis of GFP-Hepc25 in Selective pH Treatment\u003c/h2\u003e \u003cp\u003eThe stability of the peptide (800.0 \u0026micro;g/mL) under different pH conditions was evaluated by incubation at 37\u0026deg;C for 2 hours (69). For this purpose, peptide solutions were adjusted to pH 5, 7, 9, and 13, along with a control group, and incubated at 50 rpm. Following incubation, all samples were readjusted to pH 8. As a control, the peptide was incubated without any pH modification. After the incubation period, the antimicrobial activity of the peptides was assessed against microorganisms including \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e using the MIC test. This experimental design allowed for systematic evaluation of pH-dependent stability while maintaining consistent testing conditions through pH readjustment prior to antimicrobial assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGrowth Inhibition Assay of GFP-Hepc25\u003c/h2\u003e \u003cp\u003eThe growth inhibition of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e treated with GFP-Hepc25 was assessed by monitoring the absorbance at OD\u003csub\u003e600\u003c/sub\u003e nm at multiple time points. The strains were cultured in 96-well plates under conditions similar to those used for the determination of the MIC test. Various concentrations of GFP-Hepc25 were tested, and the bacterial growth was measured at every 5 hours (0, 5, 10, 15, 20, and 24) hours. The absorbance at OD\u003csub\u003e600\u003c/sub\u003e nm was recorded using a spectrophotometer. The differences in absorbance at each time point were used to determine the antimicrobial effect, revealing the dynamics of bacterial growth inhibition in response to GFP-Hepc25 (Luo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMorphological Alteration of Microorganisms\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e pre-culture was sub-cultured into a fresh LB broth medium. When the OD\u003csub\u003e600\u003c/sub\u003e nm reached 0.6, it was diluted 1:100 (Zhao et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In a sterile Falcon tube, 1 mL of 10⁶ CFU/mL bacterial culture was treated with 1 mL LB broth and 1 mL of GFP-Hepc25 (800.0 \u0026micro;g/mL) and incubated at 150 rpm, 37\u0026deg;C for 2 hours. In the control group, PBS was added instead of GFP-Hepc25. The samples were then centrifuged at 3000 xg for 5 minutes to pellet the bacteria and washed with PBS. Next, a 2.5% glutaraldehyde solution was added, and the samples were incubated overnight at +\u0026thinsp;4\u0026deg;C. The bacteria were washed with 0.1% PBS and subjected to serial ethanol washes. Each wash step was performed for 10 minutes. Ethanol was applied in increasing concentrations: 30%, 50%, 70%, 90%, 100%, and then again 100% in order to progressively dehydrate the samples and prepare them for electron microscopy analysis. Finally, the samples were placed on sticky carbon tape, dried, and observed under a scanning electron microscope (SEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm Formation Assay\u003c/h2\u003e \u003cp\u003eThe biofilm inhibition test is an analytical method used to evaluate the degree to which an agent or condition prevents microorganisms from forming biofilms on surfaces (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eO'Toole 2011\u003c/span\u003e). \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e are pathogenic bacteria with strong biofilm-forming capabilities and are commonly used in biofilm inhibition tests as they represent both Gram-positive and Gram-negative strains. The biofilm inhibition activity of GFP-Hepc25 peptide against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e was determined using the crystal violet method in 96-well plates. \u003cem\u003eS. aureus\u003c/em\u003e was incubated in Tryptic Soy Broth (TSB) medium (g/L: 17 g pancreatic casein, 2.5 g glucose, 3 g papaic soybean, 2.5 g dextrose, 5 g sodium chloride, 2.5 g dipotassium phosphate) with 0.2% glucose addition at 37\u0026deg;C for 16\u0026ndash;24 hours. \u003cem\u003eP. aeruginosa\u003c/em\u003e was incubated in Nutrient-Rich LB Broth (g/L: 15.0 g tryptone, 7.5 g yeast extract, 7.5 g sodium chloride (NaCl) and then diluted 1:100 in fresh medium. In 96-well plates, 160 \u0026micro;L of microbial suspension and 20 \u0026micro;L of peptide solution at different concentrations were added. For the negative control, 160 \u0026micro;L of microorganism and 20 \u0026micro;L of medium were used, while the positive control consisted only of medium without microorganisms. Additionally, controls containing 50% and 10% bleach were tested along with the peptides, as bleach prevents biofilm formation. After 24-hour incubation at 37\u0026deg;C, the wells were first washed with PBS, then fixed with methanol and stained with 0.1% crystal violet for 30 minutes. The wells were then washed with sterile water and 200 \u0026micro;L of 95% ethanol was added. Absorbance was measured at OD\u003csub\u003e570\u003c/sub\u003e nm. Biofilms, which are sticky structures formed by microorganisms on surfaces, were analyzed by staining with crystal violet to assess biofilm presence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Test\u003c/h2\u003e \u003cp\u003eThe L929 cell line was obtained from the American Type Culture Collection (NCTC clone 929: CCL-1). These cell lines were cultured and expanded in cell growth medium containing 10% Fetal Calf Serum (FCS), 1.0% penicillin/streptomycin (P/S), and 1% GlutaMAX. Cells were detached using 0.25% EDTA/trypsin and pelleted by centrifugation at 1,200 rpm for 4 minutes. After resuspension in fresh cell growth medium, the cells were passaged by seeding onto new cell culture plates. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) and grown in a cell culture incubator at 37\u0026deg;C with 5% CO₂ atmosphere. These prepared cells were then subjected to various experimental procedures involving specified samples and reagents for conducting cytotoxicity tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMTT Assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the MTT assay (Riss et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). L929 cells were seeded in a 96-well plate at a density of 10,000 cells per well and incubated for 24 hours at 37\u0026deg;C with 5% CO₂. Following incubation, the cell culture medium was replaced with fresh medium containing varying concentrations of the test compound. After exposure periods of 24, 48, and 72 hours, the test medium was aspirated, and 110 \u0026micro;L of MTT solution (5.0 mg/mL in PBS, 10% v/v) was added to each well, followed by a 4-hour incubation. The formazan crystals were solubilized by adding 100 \u0026micro;L of SDS solution (1% w/v in 0.01 M PBS, pH 7.4) to each well, with an additional 24-hour incubation period. The absorbance of the formazan product was then measured at OD\u003csub\u003e570\u003c/sub\u003e nm and OD\u003csub\u003e630\u003c/sub\u003e nm, and the results were calculated and compared to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using statistical methods based on results from at least three independent experiments, and the findings were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Student\u0026rsquo;s t-test and Linear Regression were applied to assess differences between groups. Results with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Additionally, to provide a precise analysis of antimicrobial activity, MIC (Minimum Inhibitory Concentration) values were determined using the Gompertz model.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003eGFP-Hepc25 and Hepc25 alone have matching performance and antimicrobial potency\u003c/h2\u003e\n \u003cp\u003eGFP-Hepc25 has demonstrated significant antimicrobial activity in performance studies conducted so far. To determine if this activity originates from the GFP tag or the Hepc25 itself, both GFP-fused and Hepc25-alone peptides were subjected to antimicrobial activity assays (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Accordingly, the antimicrobial activity of GFP-Hepc25 and Hepc25 alone were confirmed against both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Calculations revealed that the MIC of GFP-Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e was 258.264 \u0026micro;g/mL, while the MIC against \u003cem\u003eS. aureus\u003c/em\u003e was 377.74 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Otherwise, the MIC of cut Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e was 279.84 \u0026micro;g/mL, while the MIC against \u003cem\u003eS. aureus\u003c/em\u003e was 238.74 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). These MIC values represent the minimum concentration of hepcidin required to effectively inhibit microbial growth. The calculated MIC values are provided in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e in detail. There is no significant difference in antimicrobial activity between GFP-Hepc25 and Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e. However, for \u003cem\u003eS. aureus\u003c/em\u003e, the MIC value of the GFP-Hepc25 is 377.74 \u0026micro;g/mL, while the MIC for the non-GFP-tagged peptide is 238.74 \u0026micro;g/mL. The higher MIC for the GFP-Hepc25 suggests lower antimicrobial activity compared to the Hepc25. Conversely, the lower MIC for Hepc25 indicates greater antimicrobial efficacy. Although the difference in MIC values between the two forms of hepcidin for \u003cem\u003eS. aureus\u003c/em\u003e is relatively small, these results suggest that GFP itself does not exhibit toxic effects, which is in line with the literature knowledge related to its active use for gene expression in mammalian cells of GFP (Alexander et al. \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e). This observation supports the conclusion that GFP tagging does not compromise the antimicrobial potential of the hepcidin peptide; instead stands out as a highly efficient step in the solubilization of hepcidin and easy recombinant production and purification process (R\u0026uuml;cker et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e;Waldo et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eHere, we propose highly yielded and cost-effective recombinant GFP-Hepc25 complex production and purification path. Well-known that recovery of recombinant proteins from bacterial systems often demands meticulous fine-tuning of expression and purification conditions. To significantly simplify this process, GFP was integrated into bacterial expression vectors. GFP not only improves folding efficiency and solubility (R\u0026uuml;cker et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e; Alfasi et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e) but also streamlines production and purification workflows through highly refined methodologies. Moreover, this fusion strategy facilitates the precise detection of GFP-tagged hybrid proteins in intact bacterial cells via fluorescence microscopy (Venkatesh et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). To efficiently evaluate a range of conditions critical for protein expression, GFP fluorescence, which reflects expression levels, was directly measured in live bacterial cultures using a fluorescence plate reader (Chachami et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). This approach enables real-time monitoring of protein expression (Venkatesh et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e) (Fig. S4 and S6) and suggests that GFP tagging enhances protein stability and solubility (R\u0026uuml;cker et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e) (Fig S5 and S7, and S8) instead traditional approach (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). Furthermore, our GFP-fused Hepc25 exhibits antimicrobial activity comparable to or exceeding that of Hepc25 alone (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), aligning with findings from numerous studies (Maisetta et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lombardi et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Notably, Hepc25 demonstrates greater antimicrobial activity at acidic pH (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) (Maisetta et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Maisetta et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), stressing the potential of GFP-Hepc25 as a strategic framework for developing advanced therapeutics aimed at infections localized in physiologically acidic environments. Integrating the GFP tag enables precise real-time monitoring, further enhancing its applicability. This approach positions engineered Hepc25-derived peptides as a cutting-edge solution for combating a broad spectrum of infections across various anatomical niches and pathological conditions. However, challenges such as the complex synthesis required to establish disulfide bridges critical for antimicrobial activity (Shike et al. \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e), partial inhibition by body fluids (Maisetta et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e), and the relatively high concentrations needed for activity pose significant barriers to the clinical application of hepcidin-derived peptides. These limitations underscore the necessity of further experimental research to optimize these peptides for therapeutic use. Alternatively, a more cost-effective and efficient approach may involve the recombinant production of Hepc25 fused with a GFP tag, presenting promising opportunities for developing a novel GFP-Hepc25 complex as a potential antimicrobial peptide, maybe offering an innovative alternative to next-generation antibiotics.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eSelective optimizations of recombinant GFP-Hepc25 made cost-efficient paradigm\u003c/h2\u003e\n \u003cp\u003eOnce the successful transformation of the GFP-Hepc25 sequence that was cloned into the pET28(a) vector, test expression revealed substantial protein accumulation in the cytosol of the host cells (Fig. S3). These results are consistent with findings reported in the literature (Sadr et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Initially, media optimization was conducted to identify the most efficient medium for experimental procedures. The experiment was performed on a microscale using the BioLector microbioreactor. The media were ranked according to fluorescence intensity measured in a bioreactor, from highest to lowest, nu: 2\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026thinsp;\u0026gt;\u0026thinsp;11\u0026thinsp;\u0026gt;\u0026thinsp;3\u0026thinsp;\u0026gt;\u0026thinsp;4\u0026thinsp;\u0026gt;\u0026thinsp;6\u0026thinsp;\u0026gt;\u0026thinsp;8\u0026thinsp;\u0026gt;\u0026thinsp;9\u0026thinsp;\u0026gt;\u0026thinsp;7\u0026thinsp;\u0026gt;\u0026thinsp;5\u0026thinsp;\u0026gt;\u0026thinsp;10. This ranking indicates that the second composition (Casein 10 g/L, Yeast Extract 10 g/L, NaCl 10 g/L) yielded the highest fluorescence intensity, reflecting the most efficient Hepc25 production, while other media showed progressively lower performance (Fig. S4). The fluorescent capability of GFP (Chachami et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) allowed real-time monitoring of protein intensity during BioLector screening, offering that GFP-tagged protein production is not only advantageous for improving protein solubility but also serves as a valuable method for real-time tracking and analysis. This is in line with our SDS-PAGE result (Fig. S5). Once the max media optimization, cells containing the GFP-Hepc25 complex were screened at the micro-scale with different IPTG values (0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 1.0 mM) to find the most efficient induction value (Fig. S6). Accordingly, at the end of the incubation in the BioLector microbioreactor, bacterial growth curves and fluorescence measurements at 436/540 nm (excitation/emission) wavelengths were analyzed to assess GFP-tag expression. The bacterial growth curve showed an increase in growth across all samples except for the positive control The positive control consisted of a bacteria-free medium, while the negative control was a bacterial strain not induced with IPTG. The GFP-tag expression graph indicated an increased trend in fluorescence for all samples, excluding the positive and negative controls. The time-dependent growth curve of the \u003cem\u003eE. coli\u003c/em\u003e Rosetta (DE3) was monitored using a BioLector. By analyzing the time-dependent fluorescence measurements at 436/540 nm (excitation/emission) wavelengths, it was observed that induction with varying concentrations of IPTG successfully enhanced the protein\u0026apos;s expression levels. Induction was successful at all IPTG concentrations, with increasing fluorescence intensity indicating a corresponding rise in GFP-Hepc25 expression. In contrast, no increase in expression was observed in the negative control, which was not induced with IPTG. The results demonstrate effective induction and protein expression across all IPTG concentrations tested, ranging from 0.1 mM to 1 mM. This agrees with our SDS-PAGE analysis (Fig. S7). The thickness and intensity of the bands in the gel correspond to the protein expression levels, which increased with IPTG concentration. In the negative control, which was not induced with IPTG, no discernible band was observed. These findings confirm, consistent with the fluorescence analysis, that protein expression was successfully induced in a concentration-dependent manner. Based on this result and literature, we prefer to use 0.1 mM IPTG amount in further experimental setup to provide cost-effective recombinant production (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSilaban 2018\u003c/span\u003e). Reducing the concentration of IPTG, a widely used inducer in recombinant protein production, offers significant economic and industrial advantages (Larentis et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). IPTG is a costly chemical, and its reduced usage can substantially lower production expenses, particularly in large-scale industrial applications. Utilizing low IPTG concentrations while maintaining efficient protein yields minimizes process costs, making production more economically viable (Lecina et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Additionally, lower IPTG levels help reduce cell stress, ensuring better cell health and consistent protein production, which is crucial for industrial-scale operations (Olaofe et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e;Lecina et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Post-induction temperature is a critical parameter in proteins with IPTG added since the temperature following IPTG induction significantly influences the proper folding and solubility of recombinant proteins (Huang et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lower temperatures (typically 16\u0026ndash;25\u0026deg;C) slow down protein synthesis, reducing misfolding and the formation of insoluble inclusion bodies (Chalmers et al. \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSilaban 2018\u003c/span\u003e; Mohammadpour-Aghdam et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Otherwise, induction at higher temperatures (30\u0026ndash;37\u0026deg;C) often results in rapid protein synthesis, which can lead to misfolding and loss of function. In contrast (M\u0026uuml;hlmann et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Accordingly, the post-induction temperature was optimized along with the effective IPTG concentration of 0.1 mM, and experimental results 436/540 nm (excitation/emission) of fluorescent emission for 18\u0026deg;C, 30\u0026deg;C, and 37\u0026deg;C are 4.6, 4.3, and 3.8 values, respectively) demonstrated that the most efficient temperature was 18\u0026deg;C (Fig. S8), which is consistent with the literature (Chalmers et al. \u003cspan class=\"CitationRef\"\u003e1990\u003c/span\u003e; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSilaban 2018\u003c/span\u003e; Mohammadpour-Aghdam et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eUncut GFP-Hepc25 complex offers a stable antimicrobial framework\u003c/h2\u003e\n \u003cp\u003eOnce well-optimization of the GFP-Hepc25, various performance tests were performed to assess its structural integrity and biological activity in various applications. Heat can denature AMPs, leading to the loss of their antimicrobial efficacy by disrupting secondary or tertiary structures (Phambu et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The stability of the GFP-Hepc25 was examined after exposure to 100\u0026deg;C for 5, 10, and 30-mins intervals. Accordingly, no degradation in stability was observed for GFP-Hepc25 heated for 5 minutes, and 10 minutes at 100\u0026deg;C or those kept at 37\u0026deg;C. However, the peptides exposed to 100\u0026deg;C for 30 minutes showed aggregation, indicating a loss of stability (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). which is in line with the previously available study performed on the synthetic peptide (Fig. S9)(Dandurand et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). This is supported by the molecular dynamics (MD) simulation that was performed at constant temperatures (40\u0026deg;C and 100\u0026deg;C), suggesting AMPs could lose stability at 100\u0026deg;C having irregular and high fluctuations in RMSD plot during MD simulations at 100\u0026deg;C (Tanhaeian et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe stability of AMPs under different heavy metal salt treatments is investigated to evaluate their structural and functional stability in environments contaminated with heavy metals. Heavy metals can interact with AMPs, potentially altering their secondary structure, folding, or charge distribution, which may compromise their antimicrobial activity (Ma et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The stability of the GFP-Hepc25 under different heavy metal salt treatments against \u003cem\u003eE. coli\u003c/em\u003e was evaluated, and the MIC values were determined (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-F). The MIC values for GFP-Hepc25 were measured as 301,07 \u0026micro;g/mL at 37\u0026deg;C and under treatment with MnCl₂, ZnSO₄, MgSO₄, FeSO₄, and CuSO₄. For \u003cem\u003eE. coli\u003c/em\u003e, the MIC values were as follows: 285,26 \u0026micro;g/mL, 270,436 \u0026micro;g/mL, 269,11 \u0026micro;g/mL, 399,84 \u0026micro;g/mL, 349,52 \u0026micro;g/mL. These findings indicate that heavy metal salts did not compromise the structural integrity of GFP-Hepc25 or its antimicrobial activity. Furthermore, the relatively higher MIC observed in the presence of FeSO₄ and CuSO₄ may suggest potential interactions between these metals and GFP-Hepc25 due to the capability of binding Cu\u0026sup2;+ of hepcidin through the amino-terminal copper\u0026ndash;nickel binding motif (Kulprachakarn et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Overall, heavy metals did not induce degradation of GFP-Hepc25. Likewise, the stability of the GFP-Hepc25 against \u003cem\u003eS. aureus\u003c/em\u003e was also evaluated under various heavy metal salt treatments, and the MIC values were determined (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG-L). The results are as follows: (i) the MIC value at 37\u0026deg;C was 379,1 \u0026micro;g/mL. (ii) Under treatment with MnCl₂, the MIC value was 296,99 \u0026micro;g/mL, while ZnSO₄ resulted in a MIC of 227,086 \u0026micro;g/mL, indicating enhanced antimicrobial activity in the presence of these salts, which is an inverse correlation of the report in the literature that Mn exposure causes decreased expressions of hepcidin (Fan et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Yet, this is in line with the findings that hepcidin attenuates zinc efflux in cells (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHennigar et al. 2016\u003c/span\u003e). (iii) MgSO₄ treatments resulted in MIC values of 397,46 \u0026micro;g/mL, suggesting minimal interaction with GFP-Hepc25. Interestingly, (iv) FeSO₄ treatment showed a strong max MIC value of 404,26 \u0026micro;g/mL compared to that of other metals, indicating the effect of Fe on GFP-Hepc25 activity, consistent with studies suggesting that the hepatic peptide hepcidin regulates iron absorption, plasma concentration, and tissue distribution (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGanz and Nemeth 2013\u003c/span\u003e). CuSO₄ treatment resulted in a MIC value of 355,64 \u0026micro;g/mL, showing no degradation or loss of activity in the presence of this salt. Collectively, the findings suggest that while GFP-Hepc25 maintains its structural stability and antimicrobial activity under most heavy metal conditions, the presence of ZnSO₄, MnCl₂, and the strong max MIC value of FeSO₄ highlights potential interactions that may enhance its effectiveness. Importantly, no significant degradation of GFP-Hepc25 was observed under any of the tested conditions.\u003c/p\u003e\n \u003cp\u003eLikewise, we also performed stability analysis of GFP-Hepc25 using various chemical treatment to evaluate its structural integrity and functional resilience in acidic environments. The stability of GFP-Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e was tested with the following MIC values: 37\u0026deg;C: 297,636 \u0026micro;g/mL; sodium sulfite: 244.086 \u0026micro;g/mL; benzoic acid: 274.142 \u0026micro;g/mL; sodium benzoate: 386.92 \u0026micro;g/mL; sodium chloride: 351,9 \u0026micro;g/mL; acetic acid: 289,986 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). Similarly, the stability of GFP-Hepc25 against \u003cem\u003eS. aureus\u003c/em\u003e was evaluated under acid treatment, yielding the following MIC values: 37\u0026deg;C: 387.6 \u0026micro;g/mL; sodium sulfite: 330.004 \u0026micro;g/mL; benzoic acid: 404.94 \u0026micro;g/mL; sodium benzoate: 407.66 \u0026micro;g/mL; sodium chloride: 366,52 \u0026micro;g/mL; acetic acid: 264,452 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG-L). These results show that GFP-Hepc25 remains stable and active under acidic conditions, with enhanced antimicrobial activity in the presence of sodium chloride and acetic acid, well known that the bactericidal activity of Hepc25 increases significantly at acidic pH (Maisetta et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lombardi et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Suggests high antimicrobial activity might be related to the isolated folding yields of human hepcidin under acidic polymer-supported conditions were superior to those under basic folding conditions (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). No degradation was observed, which is correlated with the knowledge that hepcidin precipitation is also prevented under acidic conditions (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe stability and antimicrobial activity of the GFP-Hepc25 peptide under detergent treatment were evaluated against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus.\u003c/em\u003e In the tests performed on \u003cem\u003eE. coli\u003c/em\u003e, the stability of GFP-Hepc25 was investigated under different detergent conditions, and MIC values were obtained as follows: 37\u0026deg;C: 265.2 \u0026micro;g/mL, SDS: 24.14 \u0026micro;g/mL, Tween 20: 190 \u0026micro;g/mL, Triton X-100: 174.4 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Similarly, in the tests conducted on \u003cem\u003eS. aureus\u003c/em\u003e, the stability of GFP-Hepc25 under detergent treatment was assessed, and the following MIC values were measured: 37\u0026deg;C: 269 \u0026micro;g/mL, SDS: 510 \u0026micro;g/mL, Tween 20: 285.6 \u0026micro;g/mL, Triton X-100: 74.8 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-H). This indicates that detergents may disrupt the stability of peptides and inhibit their biological activity. The data were analyzed using linear regression analysis. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. For SDS, the p-value was \u0026gt;\u0026thinsp;0.05, which was not statistically significant.\u003c/p\u003e\n \u003cp\u003eWe also performed growth inhibition assay to assess the efficacy of GFP-Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) and \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). The goal was to determine whether GFP-Hepc25 could inhibit bacterial growth in a dose- and time-dependent manner, as well as to compare its activity across different bacterial species. According to the data, the inhibitory effect began at the 5-hour time point, slowing bacterial growth. When compared to the negative control of \u003cem\u003eE. coli\u003c/em\u003e, the peptide at a concentration of 500.0 \u0026micro;g/mL caused a significant reduction in growth rate (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Similarly, decreasing inhibitory effects were observed at concentrations of 250.0 \u0026micro;g/mL and 125.0 \u0026micro;g/mL compared to 500.0 \u0026micro;g/mL. These results align with the MIC value of GFP-Hepc25 against \u003cem\u003eE. coli\u003c/em\u003e (258.264 \u0026micro;g/mL). For \u003cem\u003eS. aureus\u003c/em\u003e, comparison with the negative control revealed that the peptide at 500.0 \u0026micro;g/mL also induced a marked decrease in growth rate (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Lower concentrations (250.0 \u0026micro;g/mL and 125.0 \u0026micro;g/mL) exhibited reduced inhibition compared to 500.0 \u0026micro;g/mL, consistent with the MIC value for \u003cem\u003eS. aureus\u003c/em\u003e (377.74 \u0026micro;g/mL). These findings are crucial for understanding the therapeutic potential of GFP fused Hepc25 and its possible application as a broad-spectrum recombinant antimicrobial complex (Rauf et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chachami et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Data were analyzed using linear regression analysis, with results considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n \u003cp\u003eThe SEM analyses revealed that the GFP-Hepc25 peptide exhibited significant antimicrobial effects against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). While no morphological changes were observed in the control group (without peptide) for either bacterial species, deformations in the bacterial membrane were detected in the samples containing GFP-Hepc25. Additionally, it was observed that the bacteria penetrated into one another. These deformations indicate that the peptide binds to the bacterial membrane, causing structural damage. Similar results were obtained for \u003cem\u003eS. aureus\u003c/em\u003e. By comparing the bacterial density of peptide-treated and untreated microorganisms, it was observed that the peptide exerts its antimicrobial effect through intercellular activity.\u003c/p\u003e\n \u003cp\u003eIn the biofilm inhibition assay, tests conducted against the \u003cem\u003eS. aureus\u003c/em\u003e strain showed significant biofilm inhibition at concentrations of 28 and 14 \u0026micro;M, while no inhibitory effect was detected at 9 \u0026micro;M (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). In contrast, tests performed on the \u003cem\u003eP. aeruginosa\u003c/em\u003e demonstrated measurable biofilm inhibition at all concentrations of GFP-Hepc25 (28, 14, and 9 \u0026micro;M) (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). These results indicate that GFP-Hepc25 has the potential to inhibit biofilm formation in both tested bacterial species, but the efficacy varies depending on the bacterial strain and applied concentration. The data were analyzed using the Student\u0026rsquo;s t-test, and results with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\n \u003cp\u003eThis study evaluated the cytotoxic effects of GFP-Hepc25 on the L929 fibroblast cell line (Fig. 9). The results demonstrate that GFP-Hepc25 reduces cell viability in a dose- and time-dependent manner. While no significant effect on cell viability was observed at low concentrations (5\u0026ndash;20 \u0026micro;M), a marked decrease occurred at 50 \u0026micro;M and higher concentrations. At high doses (150\u0026ndash;200 \u0026micro;M), cell death rates reached nearly maximal levels. These findings indicate that GFP-Hepc25 may impair vital cellular functions at certain dose ranges. In this context, the effects of GFP-Hepc25 on cell viability could be evaluated within the framework of iron homeostasis-related biochemical processes. Our study reveals the potential toxic effects of GFP-Hepc25 on the L929 cell line, contributing to a more detailed investigation of this molecule\u0026apos;s cellular mechanisms.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAntibiotic resistance has become a global health issue today. The declining efficacy of traditional antibiotics necessitates the development of next-generation antimicrobial agents. GFP-Hepc25 is a promising candidate in the fight against antibiotic resistance. Its broad-spectrum antimicrobial activity, effectiveness against both Gram-negative and Gram-positive bacteria, and low toxicity make this peptide suitable for pharmaceutical applications. In particular, GFP-Hepc25 can be used in cases such as infections. Additionally, its potential to inhibit biofilm formation could provide a significant advantage in treating chronic infections. In pharmaceutical applications, GFP-Hepc25 may be used in the form of topical creams. The peptide's low toxicity on human cells supports its therapeutic use. GFP-Hepc25 has significant potential for use in various industries, including food, pharmaceuticals, and agriculture, due to the broad-spectrum effects and low toxicity of antimicrobial peptides. This peptide stands out for its effectiveness against both Gram-negative and Gram-positive bacteria, high stability, and low toxicity to human cells. These properties enable GFP-Hepc25 to be widely used in industrial and medical applications. In the food industry, controlling microorganisms that cause food spoilage is of great importance. While chemical preservatives (e.g., benzoic acid, sodium benzoate) are effective, consumers' increasing demand for natural and safe alternatives makes antimicrobial peptides like GFP-Hepc25 an attractive option. GFP-Hepc25 could serve as a natural preservative alternative to traditional chemicals. Its stability in acidic environments and retention of antimicrobial activity support its use as a food preservative. Moreover, the peptide's increased activity in the presence of NaCl makes it even more advantageous for use in salty food products. The use of GFP-Hepc25 in the food industry can enhance food safety while meeting consumer demand for natural products. The agricultural sector is constantly seeking new solutions to control plant diseases and improve crop yields. GFP-Hepc25 could contribute to controlling plant diseases by being used as a fungicide and protective agent in agriculture. Its effectiveness against plant pathogens, in particular, makes this peptide suitable for use in agricultural pesticides. GFP-Hepc25 can be employed to prevent fungal infections in plants and treat existing infections. Additionally, its stability under environmental stress conditions could enhance durability in agricultural applications. The use of GFP-Hepc25 in agriculture may offer a more environmentally friendly alternative to chemical pesticides. The industrial-scale production of GFP-Hepc25 enables its widespread use. High-yield production through culture medium optimization and the use of low IPTG concentrations ensures a cost-effective manufacturing process. Furthermore, incubation at low temperatures (18\u0026deg;C) improves protein folding, increasing soluble protein yield. GFP-Hepc25's stability under various stress conditions, such as high temperatures, heavy metals, and acidic environments, facilitates its use in industrial applications. Its resistance to harsh conditions encountered in food processing and agricultural applications could allow GFP-Hepc25 to be effectively utilized in these fields. The peptide's broad-spectrum antimicrobial activity and low toxicity make it suitable for use in various sectors, including food, pharmaceuticals, and agriculture.\u003c/p\u003e \u003cp\u003eIn conclusion, this study outlines the process of producing a recombinant peptide using GFP to monitor its expression levels, optimize fermentation conditions, and evaluate its stability and bioactivity. The present work successfully designed, produced, and characterized the recombinant antimicrobial peptide GFP-Hepc25, demonstrating its potential as a novel antimicrobial agent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the University of Health Sciences BAP Coordination Office within the scope of the Master's Thesis Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are included within the article and its supplementary materials.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAchm\u0026uuml;ller, C., Kaar, W., Ahrer, K. et al. (2007). Npro fusion technology to produce proteins with authentic N termini in E. coli. \u003cem\u003eNat Methods\u003c/em\u003e, 4, 1037\u0026ndash;1043.\u003c/li\u003e\n \u003cli\u003eAlbano, C. R., Randers-Eichhorn, L., Bentley, W. E., \u0026amp; Rao, G. (1998). Green fluorescent protein as a real-time quantitative reporter of heterologous protein production. \u003cem\u003eBiotechnol Prog\u003c/em\u003e, 14(2), 351-354.\u003c/li\u003e\n \u003cli\u003eAlexander, L., Lee, H., Rosenzweig, M., Jung, J. U., \u0026amp; Desrosiers, R. C. (1997). EGFP-containing vector system that facilitates stable and transient expression assays. \u003cem\u003eBiotechniques\u003c/em\u003e, 23(1), 64-66.\u003c/li\u003e\n \u003cli\u003eAlfasi, S., Sevastsyanovich, Y., Zaffaroni, L., Griffiths, L., Hall, R., \u0026amp; Cole, J. (2011). Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins. \u003cem\u003eJ Biotechnol\u003c/em\u003e, 156(1), 11-21.\u003c/li\u003e\n \u003cli\u003eArezes, J., \u0026amp; Nemeth, E. (2015). Hepcidin and iron disorders: new biology and clinical approaches. \u003cem\u003eInt J Lab Hematol\u003c/em\u003e, 37(S1), 92-98.\u003c/li\u003e\n \u003cli\u003eCha, H. J., Dalal, N. G., Pham, M. Q., Vakharia, V. N., Rao, G., \u0026amp; Bentley, W. E. (1999). Insect larval expression process is optimized by generating fusions with green fluorescent protein. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e, 65(3), 316-324.\u003c/li\u003e\n \u003cli\u003eCha, H. J., Dalal, N. G., Vakharia, V. N., \u0026amp; Bentley, W. E. (1999). Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells. \u003cem\u003eJ Biotechnol\u003c/em\u003e, 69(1), 9-17.\u003c/li\u003e\n \u003cli\u003eCha, H. J., Pham, M. Q., Rao, G., \u0026amp; Bentley, W. E. (1997). Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e, 56(3), 239-247.\u003c/li\u003e\n \u003cli\u003eCha, H. J., Wu, C. F., Valdes, J. J., Rao, G., \u0026amp; Bentley, W. E. (2000). Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e, 67(5), 565-574.\u003c/li\u003e\n \u003cli\u003eChachami, G., Lyberopoulou, A., Kalousi, A., Paraskeva, E., Pantopoulos, K., \u0026amp; Simos, G. (2013). Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e, 435(4), 540-545.\u003c/li\u003e\n \u003cli\u003eChalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., \u0026amp; Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. \u003cem\u003eScience\u003c/em\u003e, 263(5148), 802-805.\u003c/li\u003e\n \u003cli\u003eChalmers, J. J., Kim, E., Telford, J. N., Wong, E. Y., Tacon, W. C., Shuler, M. L., \u0026amp; Wilson, D. B. (1990). Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e, 56(1), 104-111.\u003c/li\u003e\n \u003cli\u003eChoyam, S., Jain, P. M., \u0026amp; Kammara, R. (2021). Characterization of a potent new-generation antimicrobial peptide of Bacillus. \u003cem\u003eFront Microbiol\u003c/em\u003e, 12, 710741.\u003c/li\u003e\n \u003cli\u003eDandurand, J., Samouillan, V., Lacoste-Ferr\u0026eacute;, M. H., Lacabanne, C., Bochicchio, B., \u0026amp; Pepe, A. (2014). Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: Signature of the amyloid fibers. \u003cem\u003ePathol Biol\u003c/em\u003e, 62(2), 100-107.\u003c/li\u003e\n \u003cli\u003eDurdagi, S., Dağ, \u0026Ccedil;., Dogan, B., Yigin, M., Avsar, T., Buyukdag, C., ... \u0026amp; DeMirci, H. (2021). Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing. \u003cem\u003eStructure\u003c/em\u003e, 29(12), 1382-1396.\u003c/li\u003e\n \u003cli\u003eFahimirad, S., Ghaznavi-Rad, E., Abtahi, H. et al. (2021). Antimicrobial Activity, Stability and Wound Healing Performances of Chitosan Nanoparticles Loaded Recombinant LL37 Antimicrobial Peptide. \u003cem\u003eInt J Pept Res Ther\u003c/em\u003e, 27, 2505\u0026ndash;2515.\u003c/li\u003e\n \u003cli\u003eFan, Q., Zhou, Y., Yu, C., Chen, J., Shi, X., Zhang, Y., \u0026amp; Zheng, W. (2016). Cross-sectional study of expression of divalent metal transporter-1, transferrin, and hepcidin in blood of smelters who are occupationally exposed to manganese. \u003cem\u003ePeerJ\u003c/em\u003e, 4, e2413.\u003c/li\u003e\n \u003cli\u003eGagliardo, B., Faye, A., Jaouen, M., Deschemin, J. C., Canonne-Hergaux, F., Vaulont, S., \u0026amp; Sari, M. A. (2008). Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone. \u003cem\u003eFEBS J\u003c/em\u003e, 275(15), 3793-3803.\u003c/li\u003e\n \u003cli\u003eGanz, T. (2011). Hepcidin and iron regulation, 10 years later. \u003cem\u003eBlood\u003c/em\u003e, 117(17), 4425-4433.\u003c/li\u003e\n \u003cli\u003eGanz, T., \u0026amp; Nemeth, E. (2012). Hepcidin and iron homeostasis. \u003cem\u003eBiochim Biophys Acta\u003c/em\u003e, 1823(9), 1434-1443.\u003c/li\u003e\n \u003cli\u003eHennigar, S. R., \u0026amp; McClung, J. P. (2016). Hepcidin attenuates zinc efflux in Caco-2 cells. \u003cem\u003eJ Nutr\u003c/em\u003e, 146(11), 2167-2173.\u003c/li\u003e\n \u003cli\u003eHuang, C. J., Peng, H. L., Patel, A. K., Singhania, R. R., Dong, C. D., \u0026amp; Cheng, C. Y. (2021). Effects of lower temperature on expression and biochemical characteristics of HCV NS3 antigen recombinant protein. \u003cem\u003eCatalysts\u003c/em\u003e, 11(11), 1297.\u003c/li\u003e\n \u003cli\u003eKulprachakarn, K., Chen, Y. L., Kong, X. et al. (2016). Copper(II) binding properties of hepcidin. \u003cem\u003eJ Biol Inorg Chem\u003c/em\u003e, 21, 329\u0026ndash;338.\u003c/li\u003e\n \u003cli\u003eKrause, A., Neitz, S., Magert, H. J., et al. (2000). LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. \u003cem\u003eFEBS Lett\u003c/em\u003e, 480(2-3), 147-150.\u003c/li\u003e\n \u003cli\u003eLarentis, A. L., Nicolau, J. F. M. Q., Esteves, G. d. S. et al. (2014). Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. \u003cem\u003eBMC Res Notes\u003c/em\u003e, 7, 671.\u003c/li\u003e\n \u003cli\u003eLecina, M., Sarr\u0026oacute;, E., Casablancas, A., G\u0026ograve;dia, F., \u0026amp; Cair\u0026oacute;, J. J. (2013). IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions. \u003cem\u003eBiochem Eng J\u003c/em\u003e, 70, 78-83.\u003c/li\u003e\n \u003cli\u003eLombardi, L., Maisetta, G., Batoni, G., \u0026amp; Tavanti, A. (2015). Insights into the antimicrobial properties of hepcidins: advantages and drawbacks as potential therapeutic agents. \u003cem\u003eMolecules\u003c/em\u003e, 20(4), 6319-6341.\u003c/li\u003e\n \u003cli\u003eLuo, X., Song, Y., Cao, Z., Qin, Z., Dessie, W., He, N., ... \u0026amp; Tan, Y. (2022). Evaluation of the antimicrobial activities and mechanisms of synthetic antimicrobial peptide against food-borne pathogens. \u003cem\u003eFood Biosci\u003c/em\u003e, 49, 101903.\u003c/li\u003e\n \u003cli\u003eMa, X., Wang, Q., Ren, K., Xu, T., Zhang, Z., Xu, M., Rao, Z., \u0026amp; Zhang, X. (2024). A Review of Antimicrobial Peptides: Structure, Mechanism of Action, and Molecular Optimization Strategies. \u003cem\u003eFermentation\u003c/em\u003e, 10(11), 540.\u003c/li\u003e\n \u003cli\u003eMaisetta, G., Di Luca, M., Esin, S., Florio, W., Brancatisano, F. L., Bottai, D., Campa, M., \u0026amp; Batoni, G. (2008). Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3. \u003cem\u003ePeptides\u003c/em\u003e, 29(1), 1-6.\u003c/li\u003e\n \u003cli\u003eMaisetta, G., Petruzzelli, R., Brancatisano, F. L., Esin, S., Vitali, A., Campa, M., \u0026amp; Batoni, G. (2010). Antimicrobial activity of human hepcidin 20 and 25 against clinically relevant bacterial strains: effect of copper and acidic pH. \u003cem\u003ePeptides\u003c/em\u003e, 31(11), 1995-2002.\u003c/li\u003e\n \u003cli\u003eMaisetta, G., Vitali, A., Scorciapino, M. A., Rinaldi, A. C., Petruzzelli, R., Brancatisano, F. L., ... \u0026amp; Batoni, G. (2013). pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25. \u003cem\u003eFEBS J\u003c/em\u003e, 280(12), 2842-2854.\u003c/li\u003e\n \u003cli\u003eMohammadpour-Aghdam, M., Molaeirad, A., \u0026amp; Azizi, A. (2021). The Effect of DMSO, IPTG and Incubation Temperature on Growth Hormone Protein Expression in E. coli. \u003cem\u003eBiomacromol J\u003c/em\u003e, 7(3), 86-92.\u003c/li\u003e\n \u003cli\u003eM\u0026uuml;hlmann, M., Forsten, E., Noack, S. et al. (2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. \u003cem\u003eMicrob Cell Fact\u003c/em\u003e, 16, 220.\u003c/li\u003e\n \u003cli\u003eOlaofe, O. A., Burton, S. G., Cowan, D. A., \u0026amp; Harrison, S. T. (2010). Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli. \u003cem\u003eBiochem Eng J\u003c/em\u003e, 52(1), 19-24.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Toole, G. A. (2011). Microtiter dish biofilm formation assay. \u003cem\u003eJ Vis Exp\u003c/em\u003e, 47, e2437.\u003c/li\u003e\n \u003cli\u003ePhambu, N., Almarwani, B., Garcia, A. M., Hamza, N. S., Muhsen, A., Baidoo, J. E., \u0026amp; Sunda-Meya, A. (2017). Chain length effect on the structure and stability of antimicrobial peptides of the (RW)n series. \u003cem\u003eBiophys Chem\u003c/em\u003e, 227, 8-13.\u003c/li\u003e\n \u003cli\u003ePrasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., \u0026amp; Cormier, M. J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. \u003cem\u003eGene\u003c/em\u003e, 111(2), 229-233.\u003c/li\u003e\n \u003cli\u003eRauf, A., Shariati, M. A., Khalil, A. A., Bawazeer, S., Heydari, M., Plygun, S., ... \u0026amp; Aljohani, A. S. (2020). Hepcidin, an overview of biochemical and clinical properties. \u003cem\u003eSteroids\u003c/em\u003e, 160, 108661.\u003c/li\u003e\n \u003cli\u003eRiss, T. L., et al. (2013). Cell Viability Assays. Eli Lilly \u0026amp; Company and the National Center for Advancing Translational Sciences.\u003c/li\u003e\n \u003cli\u003eRochette, R., Gudjoncik, A., Guenancia, C., Zeller, M., Cottin, Y., \u0026amp; Vergely, C. (2015). The iron-regulator hormone hepcidin: a possible therapeutic target? \u003cem\u003ePharmacol Ther\u003c/em\u003e, 146, 35-52.\u003c/li\u003e\n \u003cli\u003eR\u0026uuml;cker, E., Schneider, G., Steinh\u0026auml;user, K., L\u0026ouml;wer, R., Hauber, J., \u0026amp; Stauber, R. H. (2001). Rapid evaluation and optimization of recombinant protein production using GFP tagging. \u003cem\u003eProtein Expr Purif\u003c/em\u003e, 21(1), 220-223.\u003c/li\u003e\n \u003cli\u003eSadr, V., Saffar, B., \u0026amp; Emamzadeh, R. (2017). Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology. \u003cem\u003eGene\u003c/em\u003e, 610, 112-117.\u003c/li\u003e\n \u003cli\u003eShike, H., Lauth, X., Westerman, M. E., Ostland, V. E., Carlberg, J. M., Van Olst, J. C., Shimizu, C., Bulet, P., \u0026amp; Burns, J. C. (2002). Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. \u003cem\u003eEur J Biochem\u003c/em\u003e, 269(8), 2232-2237.\u003c/li\u003e\n \u003cli\u003eShimomura, O., Johnson, F. H., \u0026amp; Saiga, Y. (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. \u003cem\u003eJ Cell Comp Physiol\u003c/em\u003e, 59(3), 223-239.\u003c/li\u003e\n \u003cli\u003eSilaban et al. (2018). IOP Conf. Ser.: Earth Environ. Sci., 217, 012039.\u003c/li\u003e\n \u003cli\u003eTanhaeian, A., Mohammadi, E., Mansury, D., \u0026amp; Zeinali, T. (2020). Assessment of a novel antimicrobial peptide against clinically isolated animal pathogens and prediction of its thermal-stability. \u003cem\u003eMicrob Drug Resist\u003c/em\u003e, 26(4), 412-419.\u003c/li\u003e\n \u003cli\u003eVenkatesh, B., Arifuzzaman, M., Mori, H., Suzuki, S., Taguchi, T., \u0026amp; Ohmiya, Y. (2005). Use of GFP tags to monitor localization of different luciferases in E. coli. \u003cem\u003ePhotochem Photobiol Sci\u003c/em\u003e, 4(9), 740-743.\u003c/li\u003e\n \u003cli\u003eWaldo, G., Standish, B., Berendzen, J. et al. (1999). Rapid protein-folding assay using green fluorescent protein. \u003cem\u003eNat Biotechnol\u003c/em\u003e, 17, 691\u0026ndash;695.\u003c/li\u003e\n \u003cli\u003eWiegand, I., Hilpert, K., \u0026amp; Hancock, R. E. W. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. \u003cem\u003eNat Protoc\u003c/em\u003e, 3(2), 163-175.\u003c/li\u003e\n \u003cli\u003eWinterbourn, C. C. (1995). Toxicity of iron and hydrogen peroxide: the Fenton reaction. \u003cem\u003eToxicol Lett\u003c/em\u003e, 82-83, 969-974.\u003c/li\u003e\n \u003cli\u003eZhang, H., Yuan, Q., Zhu, Y., \u0026amp; Ma, R. (2005). Expression and preparation of recombinant hepcidin in Escherichia coli. \u003cem\u003eProtein Expr Purif\u003c/em\u003e, 41(2), 409-416.\u003c/li\u003e\n \u003cli\u003eZhang, J., Diamond, S., Arvedson, T., Sasu, B. J., \u0026amp; Miranda, L. P. (2010). Oxidative folding of hepcidin at acidic pH. \u003cem\u003eBiopolymers\u003c/em\u003e, 94(2), 257-264.\u003c/li\u003e\n \u003cli\u003eZhao, F., Yang, N., Wang, X., Mao, R., Hao, Y., Li, Z., ... \u0026amp; Wang, J. (2019). In vitro/vivo mechanism of action of MP1102 with low/nonresistance against Streptococcus suis type 2 strain CVCC 3928. \u003cem\u003eFront Cell Infect Microbiol\u003c/em\u003e, 9, 48.\u003c/li\u003e\n \u003cli\u003eZuo, X., Mattern, M. R., Tan, R., Li, S., Hall, J., Sterner, D. E., Shoo, J., Tran, H., Lim, P., Sarafianos, S. G., Kazi, L., Navas-Martin, S., Weiss, S. R., \u0026amp; Butt, T. R. (2005). Expression and purification of SARS coronavirus proteins using SUMO-fusions. \u003cem\u003eProtein Expr Purif\u003c/em\u003e, 42(1), 100-110.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial peptide, GFP-Hepc25, Hepc25, Fermentation optimization, Peptide Stability, Minimum inhibitory Concentration","lastPublishedDoi":"10.21203/rs.3.rs-6555765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6555765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial peptides (AMPs) are natural compounds with broad-spectrum activity, playing a key role in the innate immune system by disrupting pathogen membranes. This study evaluates the recombinant antimicrobial peptide GFP-Hepc25, where GFP tagging facilitates fluorescence monitoring without additional staining. Optimal growth conditions for GFP-Hepc25 expression were determined as 18\u0026deg;C, IPTG concentrations of 0.1\u0026ndash;1 mM, and a casein-based medium with yeast extract and NaCl. Both GFP-Hepc25 and Hepc25 demonstrated antimicrobial activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. GFP-Hepc25 exhibited notable stability under heat, and acidic conditions, as well as in the presence of MnCl₂, ZnSO₄, MgSO₄, CuSO₄, and FeSO₄. It retained stability at 100\u0026deg;C for 5 and 10 minutes, though prolonged heating caused degradation. However, stability decreased under alkaline pH and with detergents such as SDS, Triton-X100, and Tween20. GFP-Hepc25 significantly inhibited \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e biofilm formation. These findings highlight GFP-Hepc25 as a promising next-generation antimicrobial peptide.\u003c/p\u003e","manuscriptTitle":"Harnessing the Power of GFP Fusion in E. coli for Well-Optimized Expression and Enhanced Characterization of Human Hepcidin-25","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 12:30:17","doi":"10.21203/rs.3.rs-6555765/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56f97c82-1c09-40fa-9770-c9509db62781","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-16T17:23:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 12:30:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6555765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6555765","identity":"rs-6555765","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}