Insulin-like growth factor 1 (IGF1) overexpression on a phycocyanin carrier protein in cyanobacteria

Insulin-like growth factor 1 (IGF1) overexpression on a phycocyanin carrier protein in cyanobacteria | 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 Insulin-like growth factor 1 (IGF1) overexpression on a phycocyanin carrier protein in cyanobacteria Bharat Kumar Majhi, Anastasios Melis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9453408/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract The Insulin-like Growth Factor 1 (IGF1) is a small (~ 7.6 kDa) protein-hormone primarily generated in the liver of mammals. It has a similar structure to insulin, and functions to support cell growth and development. As a result, IGF1 is in high demand in the biopharmaceutical, biomedical, and research fields. In eukaryotic photosynthetic systems, expression of this and other mammal proteins has been met with limited success. The current study used Synechocystis sp. PCC 6803, a freshwater, single-celled cyanobacterium, as a host system to produce the IGF1 through recombinant DNA technology. To stabilize and enhance IGF1 accumulation in the cells, fusion constructs of the codon optimized IGF1 gene with the β-subunit ( cpcB gene) of phycocyanin were designed. The resulting transgenic strains accumulated significant amounts of recombinant IGF1 in direct proportion to phycocyanin in Synechocystis . Quantitative aspects of the work, including the culture biomass yield in photoautotrophic and mixotrophic growth conditions, and the amount of recombinant fusion protein (CpcB*IGF1), as a fraction of total cellular protein in Synechocystis are reported. Further, antibody specificity is used as a tool to examine the effect of different fusion orientations between the CpcB and IGF1 proteins, needed to inform of optimal conditions for IGF1 functional properties. The work adds to the concept of cyanobacteria serving as cell factories for the renewable photosynthetic generation of biopharmaceutical proteins from sunlight, carbon dioxide and water. Fusion DNA and protein constructs Phycocyanin Recombinant protein stability Synechocystis sp. PCC 6803 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Insulin-like Growth Factors (IGFs) are a class of small hormonal proteins that are primarily generated in the liver of mammals and aid in organismal cell growth and development (Maki 2010 ; Khan et al. 2025 ). Mammals, including humans and bovines, possess two different IGFs: IGF1 and IGF2 (Poreba and Durzynska 2020 ; LeRoith et al. 2021 ). The human and bovine IGF1 genes encode identical 70 amino acid long polypeptides with a molecular weight of approximately 7.6 kDa (Vajdos et al. 2001 ; Rotwein 2017 ). The tertiary configuration of the IGF1 proteins comprise an asymmetrical helix-coil protein structure, having a catalytic site closer to its N-terminus domain (Li et al. 2019 ). Because of its importance in cell growth and development, and in addition to its use in basic research, IGF1 is in high demand as a growth supplement by the cultured meat and leather industries. This necessitates sufficient supplies to meet the growing demand. Photosynthetic organisms provide an interesting platform for recombinant IGF1 production as they do not need an external source of organic nutrients to grow (Panahi et al. 2003 ; Poudel et al. 2017 ; Park et al. 2020 ). They primarily rely on sunlight, carbon dioxide and water. In addition, the risk of contamination is significantly lower than that of a mammalian or fermentative cell system. However, producing recombinant IGF1 in plants is time-consuming, as plants grow slowly and require complex and often unstable genetic transformation protocols. Furthermore, the stability of recombinant proteins themselves in non-native organisms, especially plants and algae, is often severely compromised by intracellular enzymes and the cellular proteasome that recognize and degrade exogenous proteins (Zhang et al. 2021 ), resulting in very low steady state yields (Majhi and Melis 2025a ). Such pitfalls can be alleviated using cyanobacteria. Unicellular cyanobacteria offer a platform to produce a wide variety of bioactive chemicals (Lindberg et al. 2010 ; Singh et al. 2011 ; Formighieri and Melis 2016 ; Betterle and Melis 2019 ; Price et al. 2020 ; Singh et al. 2020 ; Santos-Merino et al. 2023 ; Melis et al. 2024 ) and recombinant proteins (Betterle et al. 2020 ; Zhang et al. 2021 ; Hidalgo Martinez et al. 2022 ; Hidalgo Martinez and Melis 2023 ; Majhi and Melis 2024 ; Melis et al. 2024 ; Majhi and Melis 2025a , b ). They grow much faster than plants and are amenable to stable genetic transformation, including spontaneous uptake of exogenous DNA and a direct double homologous recombination, leading to successful transformations. The latter enable the generation of genetically modified strains, suitable for the expression of recombinant enzymes and other proteins (Berla et al. 2013 ). The ease of uptake of exogenous DNA, combined with homologous recombination and photoautotrophic or heterotrophic growth, makes them ideal hosts for recombinant protein production (Berla et al. 2013 ). Furthermore, stability of recombinant protein expression in cyanobacteria is enhanced by the fusion of otherwise unstable recombinant proteins with highly-expressed native proteins, e.g. phycocyanin, which has been shown to substantially improve recombinant protein stability and accumulation within the cells (Zhang et al. 2021 ; Hidalgo Martinez and Melis 2023 ; Majhi and Melis 2024 ). In the present study, the cyanobacterium Synechocystis sp. PCC 6803 ( Synechocystis ) was used as a model organism and a host to test for the expression of IGF1. Four genetic fusion constructs between the phycocyanin CpcB β-subunit and the IGF1 gene were designed and successfully integrated into the Synechocystis genome for recombinant IGF1 production. Two of these constructs entailed a CpcB C-terminus fusion to the IGF1 N-terminus (forward IGF1 expression). The other two entailed a CpcB C-terminus fusion to the IGF1 C-terminus (inverse IGF1 expression). The fusion constructs of forward-IGF1 and inverse-IGF1 with the β-subunit of phycocyanin were successfully expressed and accumulated in stoichiometric amounts with the CpcB protein. The synthesized Phyco*IGF1 proteins (CpcB*forward-IGF1 and CpcB*inverse-IGF1) were effectively isolated from crude cellular extracts using differential cobalt affinity column chromatography. Moreover, the IGF1 protein was successfully cleaved and separated from the Phyco*IGF1 fusion form, upon the action of recombinant proteases. The current study also examined the effect of antibiotic and glucose on Synechocystis cell growth and biomass yield. It suggested a cost-effective and efficient method for using recombinant DNA technology to exploit cyanobacteria as photosynthetic cell factories in the production of IGF1. Materials and methods Genomic DNA constructs Genetically modified cyanobacterial strains were generated upon transformation of the wild-type Synechocystis . The highly expressed phycocyanin cpc operon in Synechocystis , consisting of cpcB , cpcA , cpcC2 , cpcC1 , and cpcD genes that code for different subunits of the abundant phycocyanin (Fig. 1 A) were the subject of this genetic transformation. The cpc operon was modified upon replacing the native cpcB gene, encoding the β-subunit of phycocyanin, with a fusion construct of the cpcB in the leading position and the IGF1 in the trailing position of the linear construct. The latter was fused in the forward (N-to-C) or inverse (C-to-N) orientation (please see below). In this arrangement, the IGF1 gene was inserted prior to the stop codon at the carboxyl terminus of the cpcB gene. Additional DNA sequences were inserted between the cpcB and the IGF1 fusion, including the 6xHis tag, a spacer (S: PAEKWAPGGS), the Tobacco Etch Virus (TEV) protease cleaving sequence (tev: ENLYFQ/G), followed by the Synechocystis codon optimized DNA sequence of the Insulin-Like Growth Factor 1 (IGF1). Upon double homologous recombination, the resulting fusion constructs cpcB*6xHis*tev*IGF1 and cpcB*6xHis*S*tev*IGF1 replaced the native cpcB gene in the Synechocystis genome (Fig. 1 B and 1 C). The transgenic strains harboring the aforementioned DNA constructs in the genome were termed Syn*tev*IGF1 and Syn*S*tev*IGF1 (Fig. 1 B,C). Two additional fusion constructs was generated by assembling DNA sequences of cpcB , a spacer (S: PAEKWAPGGS), 6xHis tag, the Human Rhinovirus 3C (HRV) protease cleaving sequence (hrv: LEVLFQ/GP), followed by the codon optimized DNA sequence of the Insulin-Like Growth Factor 1 (inv-IGF1). The resulting fusion constructs cpcB*6xHis*hrv*IGF1 and cpcB*S*6xHis*hrv*IGF1 replaced the native cpcB gene in the Synechocystis genome (Fig. 1 D,E). The transgenic strains harboring the DNA constructs were termed Syn*hrv*IGF1 and Syn*S*hrv*IGF1 (Fig. 1 D,E). The spacer S was designed with the inclusion of two prolines to change the relative orientation of the IGF1 protein relative to that of the CpcB β-subunit and thus to probe for potential hindrances in the assembly of the (α,β*IGF1) 3 G1 heterohexameric disc (please see discussion section). The cleaving sequences tev and hrv were introduced in the constructs to test for the cleaving efficiency of the IGF1 from the isolated (α,β*IGF1) 3 G1 heterohexameric Phyco*IGF1 fusion protein forms. All the DNA constructs were designed in the lab and were synthesized by Biomatik USA (Wilmington, DE, United States). The resulting corresponding plasmids were used to transform wild type Synechocystis , as described (Majhi and Melis 2024 ). All cyanobacterial strains were maintained on BG-11 agar plates containing 1% agar, 0.3% sodium thiosulfate, 10 mM TES-NaOH (pH 8.2), and appropriate antibiotics (kanamycin, 25 µg mL − 1 or chloramphenicol, 15 µg mL − 1 ). All plates and cultures were kept at 25°C under continuous illumination at 40 µmol photons m − 2 s − 1 . Genomic DNA PCR analysis To determine the genomic DNA state of transformants, colony PCR analysis was performed using cyanobacterial liquid cultures. A small aliquot of cyanobacterial culture (20 µL) was mixed with 100% ethanol (20 µL) and Chelex® 100 resin (100 µL, 10% w/v) (BioRad: 142–1253), and heated at 98 ºC for 10 min prior to centrifugation at 16,000 g for 10 min. The supernatant was used as a DNA template for the PCR analysis, as follows. In a 25 µL reaction mixture, 2.5 µL of supernatant was combined with Q5 High-Fidelity 2X Master Mix (New England Biolab, Ipswich, MA) for the PCR reaction. The manufacturer's instructions were followed to accurately set the annealing temperature for primers US Fwd : 5ˈ-ACCTGTAGAGAAGAGTCCC-3ˈ; DS_Rev : 5ˈ-GCGGAATATTGTCAACCAG-3ˈ (Fig. 1 B,C), and cpcB-Fwd : GTATTCACTCGGGTT; cpcA-Rev : GGTGCGGTTGATTTCATC (Fig. 1 D,E),). Amplified PCR products were analyzed on a 1% agarose gel to identify the DNA PCR products and thus to test the homoplasmy status of the transformants genomic DNA. Cell growth and dry cell weight measurement Cells were grown in 100 mL BG-11 liquid media until OD 730 reached 0.8-1.0. Cells were harvested by centrifugation at 8,000 g for 15 min, the supernatant was discarded, and the pellet was resuspended in 5 mL of fresh BG-11. In photoautotrophic growth cultivation, cells were inoculated to an OD 730 of 0.05 in 100 mL of BG-11 in the absence or presence of antibiotic, as a selectable marker. In mixotrophic growth, cells were grown in the presence of 5 mM glucose in addition to the above-mentioned conditions. Cultures were kept on a shaker (rpm: 100) at 30°C under continuous illumination (40 µmol photons m − 2 s − 1 ) for 7 days. The OD 730 was measured every 24 h. The rate of growth was plotted as a function of growth time, using the GraphPad Prism or KaleidaGraph software. Following 7 days of growth, cells were pelleted by centrifugation at 8,000 g for 15 min. Wet pellets were rinsed twice and dried at 90°C for approximately 2–3 h prior to dry weight measurement. Dry cell weight data were plotted using the GraphPad Prism software. Whole cell absorption spectra were measured as described (Majhi and Melis 2024 ). Cells were pelleted by centrifugation at 5,000 g for 15 min after cultures reached an OD 730 of 0.8-1.0. Pellets were resuspended in aliquots of 2 mL of BG-11 prior to measurement. A Shimadzu UV-1800 spectrophotometer was used to measure the absorbance spectra, with the latter suspended at an optical cell density OD 730 = ~ 0.4. The optical path length and scanning speed were set to 1 cm and 200 nm/min, respectively. GraphPad Prism or KaleidaGraph software were used to plot the resulting absorbance spectra. Chlorophyll a concentration was measured as described in (Kirst et al. 2014 ). Chlorophyll and carotenoid pigments were extracted from whole or broken cells using 100% methanol, as the solvent. The methanolic solution containing these pigments was used to measure the absorbance spectra. The absorbance of the methanolic extract at 663 nm was used to calculate the chlorophyll a content of the samples. Similarly, the A 625 nm value from the whole cell absorbance spectra was used to estimate the phycocyanin content of cells. Protein Isolation Cells were harvested at an OD 730 of 0.8-1.0 by centrifugation at 8,000 g for 15 min. Pellets were resuspended in 25 mM Tris-HCl, pH-8.2, solution supplemented with cOmplete™ mini protease inhibitor cocktail (2 mL/300 mL cell culture). The cell suspension was passed through a French Press three times at a pressure of 1,500 PSI to disrupt the cells. Unbroken cells were pelleted by centrifugation at 400 g for 3 min. The 6xHis-tagged proteins were isolated from the crude cellular extracts using a differential cobalt-affinity column chromatography. Prior to the His-tagged protein isolation, the crude cellular extracts were incubated in 20 mM HEPES buffer, pH-7.5, containing 0.5% Triton-X 100 at 4°C for 30 min in a spinning rotor (15 cycles/min). Following incubation, the crude cellular extracts were centrifuged at 16,000 g for 5 min to remove insoluble materials. The supernatant, containing soluble proteins, were mixed with cobalt-resin (HIS-Select® Cobalt Affinity Gel, Millipore Sigma) in a 1:1 (v:v) ratio and incubated at 4°C for 30–45 min in a spinning rotor (15 cycles/min) to facilitate protein binding to the resin. The mixture was then passed through a column and washed four times with 20 mM HEPES, pH-7.5, 150 mM NaCl, and 10 mM imidazole washing solution (10 mL/wash) to remove unbound proteins. The His-tagged fusion proteins were eluted from the Co column using 20 mM HEPES, pH-7.5, 150 mM NaCl, and 250 mM imidazole elution solution. Eluted proteins were concentrated using Amicon® Ultra 15 mL centrifugal filters (Millipore Sigma). The concentrated protein samples were snap frozen in liquid nitrogen and stored at -80°C for later use. SDS-PAGE and NATIVE-PAGE analysis The crude cell extracts were used to resolve the total Synechocystis cellular proteins. 1 µg chlorophyll a equivalent of crude cell extract was solubilized in 1x Laemmli Sample Buffer (Bio-Rad, Hercules, CA) for 30 min at room temperature. To facilitate solubilization and protein denaturation, 1 M urea and 5% β-mercaptoethanol were added to the mix, and samples were gently vortexed every 10 min. Following incubation, samples were centrifuged at 16,000 g for 3 min and the supernatant loaded onto a Precast 12-well kD™ Mini-PROTEAN® TGX™ Protein Gel (Bio-Rad, Hercules, CA) and electrophoresed at 200 V for 45 min. For the SDS-PAGE and NATIVE-PAGE analysis of cobalt column eluted protein samples, 20 µg of isolated heterohexameric phycocyanin complexes were loaded onto the gel lanes. Zinc chromophore fluorescence and Coomassie staining Zinc sulphate (ZnSO 4 ) solution was used to label the phycocyanobilin chromophore-binding proteins directly on the gel following the SDS-PAGE analysis, as follows. The electrophoresed gel was soaked in a 5 mM zinc sulphate solution for 30 min at room temperature with gentle shaking. A Chemidoc imaging system (BIORAD) was used to capture the zinc-chromophore fluorescence of the phycocyanobilin-containing protein bands, with the labeling images seen under UV light. The gel was subsequently stained in a Coomassie stain solution (0.07% brilliant blue R-250, 50% methanol, and 10% acetic acid) for 60 min at room temperature with gentle shaking to visualize and quantify specific protein bands of interest in this work. Coomassie stained gel images were taken using a Gel Doc XR+ System and Image Lab software (Bio-Rad Hercules, CA). TEV and HRV3C cleaving of the IGF1 from its carrier protein complex Cleaving the rIGF1 from the isolated (α,β*IGF1) 3 G1 Phyco*IGF1 fusion constructs was performed as follows: 20 µg of isolated (α,β*IGF1) 3 G1 harboring the Phyco*tev*IGF1 fusion protein complexes were incubated with variable concentrations (0, 20, 40, and 80 IU) of recombinant-TEV protease (rTEV) at 30°C for 3 h. In case of Phyco*hrv*IGF1 protein, 20 µg of isolated (α,β*IGF1) 3 G1 heterohexameric complexes harboring the Phyco*hrv*IGF1 fusion protein were incubated with 0 and 1 IU of recombinant-HRV 3C (rHRV) protease for 12–16 h only, at 4°C. The shorter incubation in this case was chosen as a recent study (Majhi and Melis 2025b ) showed HRV 3C to be a more efficient protease for cleaving recombinant proteins from the CpcB*P fusion constructs. Following incubation, protein samples were prepared for SDS-PAGE analysis, as described in the SDS-PAGE subsection above. Densitometry measurements of Coomassie-stained protein bands was performed using the built-in program of the Gel Doc XR+ System (Bio-Rad, Hercules, CA). Western blot analysis 20 µg of isolated (α,β*IGF1) 3 G1 heterohexameric phycocyanin protein complexes were processed as described above for SDS-PAGE and NATIVE-PAGE analysis. Following the electrophoretic separation of the (α,β*IGF1) 3 G1 constituent proteins, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and probed with anti-CpcB and anti-IGF1 specific polyclonal antibodies as described (Majhi and Melis 2024 ; Majhi and Melis 2025a ). Results Genomic DNA status of transformants The genomic DNA status of transformants was evaluated to test for the attainment of homoplasmy, meaning removal of wild type copies of DNA (Fig. 1 A) and quantitative replacement with the corresponding transgenic version harboring the heterologous fusion constructs (Fig. 1 B-E). Attaining DNA copy homoplasmy is important for determining the expression efficiency of the heterologous proteins, and the physiological and biochemical traits of genetically modified strains. A colony-PCR analysis was performed with cellular DNA as a template to test the genomic DNA status of cells. The primers used in the PCR analysis were designed to cover the modified region of the cpc operon in the genome, as well as the upstream and downstream native DNA regions of the transgenic DNA insertion. The PCR analysis of DNA from the wild type (WT), Syn*S*tev*IGF1 , and Syn*tev*IGF1 , showed a single product for each strain on agarose gel electrophoresis (Fig. 2 A). The product sizes were 1,745 base pairs (bp), and 2.929 bp, and 2,893 bp, respectively. Absence of a PCR product corresponding to the size of WT in the transformants is evidence that these cells achieved DNA copy homoplasmy. Similarly, transgenic strains Syn*hrv*IGF1 , and Syn*S*hrv*IGF1 harboring inverse-IGF1 DNA constructs cpcB*6xHis*hrv*IGF1 , and cpcB*S*6xHis*hrv*IGF1 , respectively, exhibited a single PCR product on agarose gel electrophoresis (Fig. 2 B). The product sizes of WT, Syn*hrv*IGF1 , and Syn*S*hrv*IGF1 were 983 bp, 2,140 bp, and 2,170 bp, respectively. The results show that all of the above transgenic strains have attained a state of DNA homoplasmy, meaning they possessed only transformant DNA and lacked the original wild type copies. Photoautotrophic growth Cell growth measurements are important to determine cell fitness and to assess the effect of genetic modifications on cells' ability to conduct photosynthesis and growth. The photoautotrophic growth analysis revealed that all cell types were able to use light for photosynthesis. However, the rate of growth was slower in the transformants than in the WT (Fig. 3 ). The photoautotrophic growth doubling time for the Syn*tev*IGF1 and Syn*S*tev*IGF1 strains was 38.5 ± 4.5 h, which is approximately double that of the WT strain (24 h) (Fig. 3 A). Furthermore, the transformant cultures had a more greenish coloration, compared with the WT blue green (Fig. 3 B). The slower growth and altered coloration are attributed to partial loss of phycocyanin in the transformants, as compared to that in the WT (please see below). This partial loss of the phycocyanin light-harvesting antenna is also the reason for the slower growth of the transformants under the 40 µmol photons m − 2 s − 1 sub-saturating intensity of irradiance employed (Kirst et al. 2014 ; Formighieri and Melis 2015 ). The notion of a truncated phycocyanin antenna in the transformants resulting in slower rates of light absorption and slower photosynthesis and growth under these conditions is further supported by the even slower rate of growth of the Δcpc strain in which the entire cpc operon has been deleted and, thus, there is no phycocyanin (Fig. 3 A, Δcpc ). The presence (+ A) or absence (-A) of the antibiotic selectable marker (chloramphenicol 15 µg mL − 1 ) in the growth medium was investigated in this portion of the work. Photoautotrophic growth analysis of transgenic strains containing the inverse-IGF1 DNA constructs in the absence of antibiotic exhibited a slower rate of growth compared to the WT (Fig. 3 C). The doubling time for the WT was 24 h, whereas the doubling times of the Syn*hrv*IGF1 and Syn*S*hrv*IGF1 were closer to 27 ± 1 h for the Syn*hrv*IGF1-A and Syn*S*hrv*IGF1-A strains. Interestingly, the presence of the selectable marked antibiotic (chloramphenicol) in the growth medium of the transformants (Fig. 3 C, Syn*hrv*IGF1 + A and Syn*S*hrv*IGF1 + A ) caused a further slowdown in growth to 37.5 ± 0.5 h (Fig. 3 C). Slower photoautotrophic growth in the presence of the antibiotic chloramphenicol may be linked to the extra metabolic load for the synthesis and accumulation of the chloramphenicol resistance cassette in the cells, and possibly due to adverse reactive oxygen species (ROS) production that cause damage to the photosynthetic apparatus. Chloramphenicol has been reported to promote ROS production under illumination conditions (Kodru et al. 2020 ). In this respect, cells growing in the presence of antibiotic appeared to have a lighter green coloration, compared to cells grown without antibiotic (Fig. 3 D), consistent with the above-mentioned growth mitigating conditions. Mixotrophic growth Cell growth was also measured in the presence of 5 mM glucose, with or without antibiotic, to test for the effect of an external carbon source on cell doubling time. In this case, the presence glucose enhanced the rate of growth of all strains and mitigated the effect of antibiotic that was observed under photoautotrophic conditions. (Fig. 4 A). The doubling time for WT was lowered to 17 h, that of the Syn*hrv*IGF1 + A and Syn*S*hrv*IGF1 + A strains to 20.5 ± 0.5 h, whereas in the absence of antibiotic Syn*hrv*IGF1-A and Syn*S*hrv*IGF1-A strains exhibited a faster growth of 18.5 ± 0.5 h. Faster growth in the presence 5 mM glucose is linked to the supplementary cellular activities, fueled in this case by both photosynthesis and glucose metabolism. Consistent with this notion is the coloration of the cultures, which was not adversely affected in the glucose-assisted cultivation (Fig. 4 B). Biomass yield Biomass accumulation is also an important variable in synthetic biology as it is linked to constitutive bioproducts yield. In this study, experiments were conducted to measure the dry cell weight (dcw) of all strains employed, including WT and transgenics harboring the IGF1 protein in different fusion construct configurations. Under photoautotrophic growth conditions, WT cells accumulated approximately 0.66 g/L dcw (Fig. 5 A ) compared to 0.78 g/L dcw under mixotrophic conditions (Fig. 5 B). Similarly, under photoautotrophic cultivation conditions, transgenic strains Syn*hrv*IGF1-A, Syn*S*hrv*IGF1-A, Syn*hrv*IGF1 + A , and Syn*S*hrv*IGF1 + A , accumulated 0.58 g/L, 0.55 g/L, 0.38 g/L, and 0.32 g dcw per L culture, respectively (Fig. 5 A ) . Interestingly, under mixotrophic cultivation conditions such differences in were largely alleviated, resulting in greater biomass yield, on the average in the range of 0.775 ± 0.015 g dcw per L in all five strains examined (Fig. 5 B). Phycocyanin content To determine the relative amount of phycocyanin assembled in cells, absorbance spectra of whole cell suspensions were measured. The absorbance spectrum of WT cells revealed four distinct bands with peaks at 440, 490, 625, and 680 nm (Fig. 6 A,C). The absorbance peak at 440 nm is attributed to chlorophyll a , which is associated with photosystems I and II, whereas the chlorophyll-overlapping band at 490 nm is attributed to cellular carotenoids. The 625 and 680 nm bands (shown in greater resolution in Fig. 6 B,D) are attributed to phycocyanin and chlorophyll a , respectively. The absorbance spectrum on the phycocyanin-lacking ∆ cpc strain, in which the entire cpc operon has been deleted (Kirst et al. 2014 ), is also shown. In the ∆ cpc strain, dominant are the chlorophyll a and carotenoid pigments, with the former showing a distinct 680 nm band and a lower amplitude chlorophyll a satellite peak at 625 nm. All four absorbance spectra have been normalized to the absorbance maximum of chlorophyll a at 680 nm to enable comparison in the cellular composition of phycocyanin among the four strains. The absorbance spectra of transformants Syn*tev*IGF1 , Syn*S*tev*IGF1 and Syn*hrv*IGF1, Syn*S*hrv*IGF1 exhibited a lower amplitude peak at 625 nm, corresponding to phycocyanin, compared to that of WT, showing a lower phycocyanin content in these cells (Fig. 6 ). With the A 625 of the ∆ cpc strain as baseline, we calculated the phycocyanin content in the Syn*tev*IGF1 , Syn*S*tev*IGF1 transformants to be 20.1 ± 2.0% of that in the WT (Fig. 6 B). Similarly, the phycocyanin content in the Syn*hrv*IGF1, Syn*S*hrv*IGF1 strains was estimated to be equal to 17.9% ± 2.0% of that in the WT (Fig. 6 D). These results are consistent with similar findings by Hidalgo Martinez et al., 2022 , suggesting the assembly of only the proximal to the core disc of phycocyanin, with a modified the CpcB*IGF1 β-subunit, and absence of the middle and distant phycocyanin rod discs. Thus, the modified CpcB*IGF1 β-subunit with the attendant fusion construct must have caused the assembly of a truncated phycocyanin rod. Total cell protein analysis and fusion protein isolation Total cellular protein analysis was performed to test for the presence of Phyco*IGF1 fusion proteins in the various transformants. Crude cellular extracts were analyzed through SDS-PAGE Coomassie stain and zinc-phycobilin chromophore fluorescence measurements. Coomassie stain results for WT exhibited three main protein bands migrating to 55, 19, and 17 kDa corresponding to the large subunit of Rubisco (RbcL), β-subunit (CpcB), and α-subunit (CpcA) of phycocyanin, alongside other cellular proteins (Fig. 7 A). In all transgenic strains ( Syn*tev*IGF1, Syn*S*tev*IGF1, Syn*hrv*IGF1 , and Syn*S*hrv*IGF1) , protein bands corresponding to RbcL and CpcA were still observed. However, the amount CpcA was lower in the transgenics compared to WT. Furthermore, all transgenic strains lacked the 19 kDa CpcB protein band. Instead, a newly formed protein band migrating to ~ 26–28 kDa was observed. The latter originated from the CpcB*IGF1 fusion proteins, a conclusion supported by the pronounced zinc-phycobilin fluorescence labeling (Fig. 7 B). Additionally, a 23 kDa protein band was observed in the protein extracts of transgenic strains, corresponding to the chloramphenicol antibiotic cassette (CmR), which was introduced as a selection marker. Isolation of (α,β*IGF1) 3 G1 heterohexameric complexes and cleaving of IGF1 from the CpcB*IGF1 fusion To cleave the natural form of the IGF1 from the fusion proteins, recombinant constructs were designed to include a cleaving sequence ( tev : ENLYFQ/G) specific for the Tobacco Etch Virus (TEV) protease, or ( hrv : LEVLFQ/GP) human rhinovirus 3C protease, placed immediately upstream from the IGF1 DNA sequence (Fig. 1 B-E). His-tagged fusion proteins were isolated through differential cobalt-affinity column chromatography from the crude cellular extracts as heterohexameric phycocyanin (α,β*IGF1) 3 G1 discs (Hidalgo Martinez et al. 2022 ; Hidalgo Martinez and Melis 2023 ), in which α is the α-subunit of phycocyanin, β*IGF1 is the CpcB*His*(S)*tev*IGF1, or CpcB*(S)*His*hrv*IGF1 fusion protein, and CpcG1 is the proximal phycocyanin disc linker that anchors this phycocyanin disc to the allophycocyanin core cylinders (Hidalgo Martinez et al. 2022 ; Hidalgo Martinez and Melis 2023 ). The isolated heterohexameric complexes served as templates for the IGF1 cleaving reactions. In the case of the (α,β*tev*IGF1) 3 G1, the cleaving reaction mixture involved incubating a fixed amount of isolated (α,β*tev*IGF1) 3 G1 protein complexes (20 µg protein) with different concentrations (0–80 IU) of commercially available recombinant rTEV protease at 30°C for 3 h. Following incubation, reaction mixture samples were analyzed by SDS-PAGE to determine the cleaving and separation efficiency of the IGF1 protein from the fusion protein complexes. Differential Co-affinity chromatography isolates and their analysis by SDS-PAGE from the Syn*S*tev*IGF1 strain showed three main protein bands migrating to 29, 27, and 17 kDa, corresponding to the linker CpcG1, Phyco*IGF1, and CpcA polypeptides, respectively (Fig. 8 A, 0 IU ). Addition of rTEV (20, 40, and 80 IU) to the reaction mixture harboring the (α,β*IGF1) 3 G1 complex resulted in the progressively greater appearance of cleaved Phyco* (20.7 kDa) and free IGF1 (~ 10 kDa) protein bands (Fig. 8 A,B). Protease cleaving efficiency was determined from the progressive decrease in the amount of the 27 kDa Phyco*IGF1 fusion protein and the concomitant appearance of the cleaved ~ 20.7 kDa Phyco* moiety that was observed as a function of the increasing amount of rTEV employed (Fig. 8 B). Densitometry analysis of Coomassie-stained protein gels showed that the fusion protein Phyco*IGF1 decreased to 40%, 38%, and 25% of the original in the presence of 20, 40, and 80 IU of rTEV protease, respectively. Correspondingly, the amount of the cleaved Phyco* product was measured to be 60%, 70%, and 75% of the original Phyco*IGF1 in the presence of 20, 40, and 80 IU of rTEV protease, respectively (Fig. 9 A). Similarly, when (α,β*hrv*IGF1) 3 G1 heterohexameric phycocyanin complexes harboring the hrv cleaving sequence were incubated in the presence of 0 IU and 1 IU of HRV protease at 4°C for 16 h, a greater cleaving efficiency was observed on Coomassie stained SDS-PAGE. Protein samples without HRV-treatment showed three main protein bands migrating to 29, 27 and 17 kDa corresponding to CpcG1, Phyco*IGF1, and CpcA proteins (Fig. 8 C and 8 D). The HRV-treated sample showed, in addition to Phyco*IGF1 (27 kDa) and CpcA (17 kDa), two new protein bands migrating to 24 kDa, originating from the added recombinant HRV, and ~ 21 kDa, originating from the cleaved Phyco*IGF1. Densitometry analysis of protein bands from the Coomassie stained SDS-PAGE (Fig. 8 C), showed that incubation of the isolated (α,β*hrv*IGF1) 3 G1 heterohexameric phycocyanin complexes, harboring the hrv cleaving sequence, with 1 IU of HRV protease caused a decrease of Phyco*hrv*IGF1 proteins to about 12% of the original. Conversely, the cleaved Phyco* product accounted for about 88% of the original (Fig. 9 B). A side-by-side analysis of SDS-PAGE Coomassie stain, Zn-phycobilin chromophore fluorescence, and Westen blot analysis was undertaken to probe for the presence and location of Phyco*IGF1 and the cleaved IGF1 in the protease treated protein samples (Fig. 10 ). The combined analysis revolved around the HRV reaction mixture proteins Phyco*IGF1 (~ 27 kDa), rHRV (~ 24 kDa), Phyco* (~ 21 kDa), CpcA (~ 17 kDa), and IGF1 (~ 7.6 kDa), all of which were detected in the Coomassie stain (Fig. 10 A). Three of these protein bands (Phyco*IGF1, Phyco*, and CpcA were detected with the Zn-phycobilin chromophore fluorescence method (Fig. 10 B), and only two (Phyco*IGF1 and IGF1) cross reacted in the Western blot analysis, conducted with specific anti-IGF1 polyclonal antibodies (Fig. 10 C). Differential antibody access to the (α,β*tev*IGF1) 3 G1 and (α,β*hrv*IGF1) 3 G1 complexes Specific polyclonal antibodies raised against the CpcB (anti-CpcB) and IGF1 (anti-IGF1) were employed to probe the stereochemistry and peripheral surface properties of the (α,β*tev*IGF1) 3 G1 and (α,β*hrv*IGF1) 3 G1 complexes. Native-PAGE and immunoblot analysis results of the (α,β*tev*IGF1) 3 G1 and (α,β*hrv*IGF1) 3 G1 heterohexameric complexes are shown in Fig. 11 . These undissociated complexes migrated to about 150 kDa, consistent with their protein composition. The immunoblot analysis showed that both anti-CpcB and anti-IGF1 strongly cross-reacted with the (α,β*tev*IGF1) 3 G1 complex. Surprisingly, the same antibodies failed to cross-react with the (α,β*hrv*IGF1) 3 G1 complex, suggesting a substantially different stereochemistry at the periphery of this complex, one that prevented the antibodies from accessing the antigenic epitopes of either the CpcB or IGF1 proteins. This unexpected result is rationalized upon consideration of the cleaving amino acid sequence of tev versus hrv, and in view of the inverse orientation on the IGF1 in relation to the CpcB protein. More specifically, the hrv cleaving sequence contains a proline amino acid, whereas the tev sequence does not. The proline is expected to change the angle of the following IGF1 in the fusion construct, leading to a substantially different orientation in relation to the carrier heterohexamer, whereas this did not happen with the tev sequence. In addition to the proline in the hrv cleaving sequence, there is another important difference between the two fusion construct samples examined in the Native-PAGE and immunoblot of Fig. 11 . Namely, by design, there is a C-to-N forward configuration between CpcB and IGF in the (α,β*tev*IGF1)3G1 construct, but a C-to-C inverse configuration for the IGF1 in the (α,β*hrv*IGF1)3G1 construct (Fig. 12 ). It is concluded that a proline in the hrv and inverse C-to-C fusion of the IGF1 in relation to the CpcB protein in the α,β heterohexamer contributed to structural properties that underline the differential immunoblot results in Fig. 11 (please see discussion below). Discussion Structural aspects of phycocyanin Phycobilisomes are the major pigment-protein complexes found in cyanobacteria, and they are an important component of the photosynthetic light harvesting system in these microorganisms (Singh et al. 2015 ; Zheng et al. 2021 ). They comprise large, multimeric, water-soluble protein complexes peripherally associated with the cytoplasmic side of the thylakoid membrane, where they are bound to the transmembrane CP43/CP47 chlorophyll-proteins of photosystem II (Kirst et al. 2014 ; Domínguez-Martín et al. 2022 ). Functionally, the phycobilisome plays an important role in the light-reactions of photosynthesis, capturing light energy and transferring the excitation energy to photosystem II, thereby enhancing water (H 2 O) oxidation and electron transport in the thylakoid membrane. Phycobilisomes in Synechocystis possess three allophycocyanin core cylinders, and six phycocyanin-containing peripheral rods (Kirst et al. 2014 ; Domínguez-Martín et al. 2022 ). Each of the peripheral rods comprises 6 heterohexameric (α,β)₃ discs, consisting of three α-CpcA and three β-CpcB subunits stacked to form the phycocyanin rods and stabilized through their association with colorless linker polypeptides (Singh et al. 2015 ; Zheng et al. 2021 ). The peripheral rods are linked to the core cylinders via the colorless CpcG1 linker polypeptide, which is required for the structural and functional association of phycocyanin to allophycocyanin (Domínguez-Martín et al. 2022 ; Hidalgo Martinez et al. 2022 ; Hidalgo Martinez and Melis 2023 ). The CpcB and CpcA subunits of phycocyanin are the most abundant proteins in cyanobacteria (Betterle et al. 2020 ; Hidalgo Martinez and Melis 2023 ). IGF1 protein and the CpcB*IGF1 fusion folding models The IGF1 gene encodes a protein of 70 amino acids, which has a folded helix-loop secondary structure (Nickle et al. 2024 ). The mature IGF1 protein shows a non-folding short N-terminal sequence, three core α-helices and their connecting loops, and an extended C-terminal loop (Ornitz and Itoh 2001 ) (Fig. 12 ). In mammal and human physiology, the IGF1 protein binds onto the receptor IGF1R protein through multiple interacting residues, which are distributed on the N-terminal, core helix, and C-terminus of IGF1 (Li et al. 2019 ). It has been reported that both N- and C-terminal residues of IGF1 are important for proper receptor binding and eliciting the growth signal. With this in mind, we synthesized two different fusion-constructs resulting in the stable and properly folded IGF1 protein, with receptor binding residues exposed at the C-terminal or N-terminal regions of the fusion protein. Fusion construct (Phyco*tev*IGF1) was designed to link the CpcB C-terminus to the IGF1 N- terminus (Fig. 12 A), thereby exposing the IGF1 C-terminus to the medium in the periphery of the (α,β*IGF1) 3 G1 heterohexameric phycocyanin disc. Phyco*hrv*IGF1 was designed to link the CpcB C-terminus to the IGF1 C- terminus (Fig. 12 B), thereby exposing the IGF1 N-terminus to the medium in the periphery of the (α,β*IGF1) 3 G1 heterohexameric phycocyanin disc. Protein folding models showed that, in strains harboring the Phyco*IGF1 (C-to-N fusion; Fig. 13 A) or Phyco*IGF1 (C-to-C fusion, Fig. 13 B) constructs, the IGF1 protein is localized peripherally and away from the center of the assembled (α,β)₃ discs, supported by the functional association of the (α,β*IGF1)₃G1 discs with the allophycocyanin core cylinders, and from the stability of IGF1 in these transformants (Zhang et al., 2021 ). This configuration allowed the rTEV and rHRV proteases to readily access the cleaving tev (ENLYFQ/G) and hrv (LEVLFQ/GP) sequences, respectively, resulting in the release of the IGF1 protein in the reaction medium upon its separation from the Phyco*IGF1 fusion (Fig. 8 ). Additional support for the cytosolic exposure of the fusion construct to the medium is derived from the functional association of the His-tag with cobalt in the differential cobalt (α,β*IGF1)₃G1 disc association and release upon treatment with imidazole. However, structural and functional differences existed between the (α,β*tev*IGF1)₃G1 and (α,β*hrv*IGF1)₃G1 complexes, specifically as these pertain to anti-CpcB and anti-IGF1 antibody recognition of the pertinent epitopes in the (α,β*hrv*IGF1)₃G1 complex only (Fig. 11 ). These results suggest that caution should be exercised, when fusion constructs are designed, to take into consideration the alignment of the target protein in relation to the (α,β) heterohexameric career disc. The above are important considerations in synthetic biology, where high amounts of recombinant proteins properly folded for use in the biopharmaceutical sector, or catalytically active enzymes are required for enhanced pathway yield toward the synthesis of bioactive compounds and proteins. The plant and human proteins referenced in work from this lab (Melis et al. 2024 ) would not accumulate in heterologous systems, e.g., cyanobacteria, when expressed by themselves, albeit under the control of a strong promoter (Formighieri and Melis 2014 ; Zhang et al. 2021 ; Majhi and Melis 2025a ), evidently due to the cellular proteasome activity designed to degrade denatured and foreign proteins (Zhang et al. 2021 ). This recombinant protein stability problem is apparently alleviated by the fusion constructs technology. Abbreviations Chl: Chlorophyll Car: Carotenoids Phc: Phycocyanin Apc: Allophycocyanin PBS: phycobilisome cpc : The phycocyanin-encoding operon in cyanobacteria. cpcA : Gene encoding the phycocyanin α-subunit (sll1578) cpcB : Gene encoding the phycocyanin β-subunit (sll1577) cpcG1 : Gene encoding the phycocyanin-to-allophycocyanin proximal linker protein CpcG1 (slr2051) (α,β) 3 G1: Native heterohexameric phycocyanin disc with the proximal phycocyanin-to-allophycocyanin linker protein CpcG1 IGF1: Insulin like growth factor-1 (α,β*IGF1) 3 G1: Heterohexameric phycocyanin disc with a fusion construct of the CpcB β-subunit in the leading and the IGF1 in the trailing sequence position RbcL: The large subunit of Rubisco Synechocystis: Synechocystis sp. PCC 6803 S: Oligopeptide spacer (PAEKWAPGGS) rTEV: Recombinant T obacco E tch V irus protease tev: Tobacco Etch Virus amino acid cleaving sequence (ENLYFQ/G) rHRV: Recombinant Human Rhinovirus 3C proteases hrv: Human Rhinovirus amino acid cleaving sequence (LEVLFQ/GP) Phyco*tev*IGF1: the CpcB*His*(S)*tev*IGF1 construct protein. Phyco*hrv*IGF1: the CpcB*(S)*His*hrv*IGF1 construct protein. Syn*tev*IGF1 : Transformant strain harboring the cpcB*6xHis*tev*IGF1 construct. Syn*S*tev*IGF1 : Transformant strain harboring the cpcB*6xHis*S*tev*IGF1 construct. Syn*hrv*IGF1 : Transformant strain harboring the cpcB*6xHis*hrv*IGF1 construct. Syn*S*hrv*IGF1 : Transformant strain harboring the cpcB*S*6xHis*hrv*IGF1 construct. WT: wild type Declarations Author Contributions Bharat Kumar Majhi (BKM) and Anastasios Melis (AM) designed the project. BKM conducted the experimental work. AM quantified some of the results. BKM drafted the figures and wrote the first version of the article, which was edited by AM. Declaration of competing interest Authors have no competing financial interests that could have influenced the work reported in this article. Acknowledgements The research was supported by University of California Fund # 63742. Data availability All data generated or analyzed in this work are included in this published version and the associated supplementary information files. References Berla BM, Saha R, Immethun CM, Maranas CD, Moon TS, Pakrasi HB (2013) Synthetic biology of cyanobacteria: unique challenges and opportunities. Front Microbiol 4:246. doi:https://doi.org/10.3389/fmicb.2013.00246 Betterle N, Hidalgo Martinez D, Melis A (2020) Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins. Front Plant Sci 11:237. doi: https://doi.org/10.3389/fpls.2020.00237 Betterle N, Melis A (2019) Photosynthetic generation of heterologous terpenoids in cyanobacteria. 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Biochemistry 40 (37):11022-11029. doi: https://doi.org/10.1021/bi0109111 Zhang X, Betterle N, Hidalgo Martinez D, Melis A (2021) Recombinant Protein Stability in Cyanobacteria. ACS Synthetic Biology 10 (4):810-825. doi: https://doi.org/10.1021/acssynbio.0c00610 Zheng L, Zheng Z, Li X, Wang G, Zhang K, Wei P, Zhao J, Gao N (2021) Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes. Nature Communications 12 (1):5497. doi: https://doi.org/10.1038/s41467-021-25813-y Additional Declarations No competing interests reported. Supplementary Files 20260417IGF1Suppl.pdf Appendix A. 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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-9453408","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628197250,"identity":"79385bf3-d20a-47fc-9334-7905d00f01a7","order_by":0,"name":"Bharat Kumar Majhi","email":"","orcid":"","institution":"University of California","correspondingAuthor":false,"prefix":"","firstName":"Bharat","middleName":"Kumar","lastName":"Majhi","suffix":""},{"id":628197251,"identity":"a08ed49d-71d3-4b56-afd8-cedd56570819","order_by":1,"name":"Anastasios Melis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3QMQrCMBSA4RcEp9SuFaQ9gVAp6OJhWryDOAjGJS49QAXxDE6dEx60S3XuIGiXzropKFi6uRi7CeYfH/nISwB0up+sxQTMBAAFIuqBUBJSkawm8D0BwpuQ/mq5lPft0R6FKMSVg93J/c9kmEmGRlx6vT335ZqD11WSPGBIYgwik7pocAh2SnIqmLxvcBGZ5gWfHBZqklc/ZjD0LSMEJBx8V/2WajGalIOIJq4MD9ZgnZ0VJEW83uZHx6KT4nybjp1OqrjlPdK2mhyvezQWOp1O9we9AKfbVPylLCMtAAAAAElFTkSuQmCC","orcid":"","institution":"University of California","correspondingAuthor":true,"prefix":"","firstName":"Anastasios","middleName":"","lastName":"Melis","suffix":""}],"badges":[],"createdAt":"2026-04-18 01:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9453408/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9453408/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108350334,"identity":"c118bb27-0686-413e-8f2b-371eb9e24a6f","added_by":"auto","created_at":"2026-05-03 10:09:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26956,"visible":true,"origin":"","legend":"\u003cp\u003eSchematics of the native and modified \u003cem\u003ecpc\u003c/em\u003e operon in \u003cem\u003eSynechocystis\u003c/em\u003e wild-type (WT) and transformant strains. The 5’ UTR contains the ribosome-binding sequence 5'-GTAGGAGATTAATTCA-3', which is followed by the ATG start codon of the \u003cem\u003ecpcB\u003c/em\u003e gene, denoted by the forward red arrow. The orange “I” denotes the presence of an exogenous intergenic sequence, introduced prior to the chloramphenicol resistance cassette (\u003cem\u003ecmR\u003c/em\u003e), whereas “T” denotes the operon terminator at the end of the \u003cem\u003ecpcD\u003c/em\u003e gene, followed by the 3’ UTR of this operon. (\u003cstrong\u003eA\u003c/strong\u003e) The \u003cem\u003eSynechocystis\u003c/em\u003enative \u003cem\u003ecpc\u003c/em\u003e operon comprising phycocyanin-encoding \u003cem\u003ecpcB\u003c/em\u003e and \u003cem\u003ecpcA\u003c/em\u003eand linker polypeptides \u003cem\u003ecpcC2, cpcC1,\u003c/em\u003e and \u003cem\u003ecpcD\u003c/em\u003e genes presented in blue color and marked as WT. (\u003cstrong\u003eB\u003c/strong\u003e) The modified \u003cem\u003ecpc\u003c/em\u003e operon encoding CpcB, 6xHis, tobacco etch virus protease cleaving site (\u003cem\u003etev:\u003c/em\u003e ENLYFQ/G), and \u003cem\u003eSynechocystis\u003c/em\u003ecodon-optimized bovine “Insulin-like Growth Factor 1” (\u003cem\u003eIGF1\u003c/em\u003e) gene in fusion construct configuration, followed by a chloramphenicol resistance cassette (\u003cem\u003ecmR\u003c/em\u003e) in a non-fusion operon configuration, denoted as \u003cem\u003ecpcB*6xHis*tev*IGF1 (Phyco*tev*IGF1).\u003c/em\u003e (\u003cstrong\u003eC\u003c/strong\u003e) Same as in (B) with the addition of a spacer (\u003cem\u003eS\u003c/em\u003e: PAEKWAPGGS) between \u003cem\u003e6xHis\u003c/em\u003e and \u003cem\u003etev\u003c/em\u003e, denoted as \u003cem\u003ecpcB*6xHis*S*tev*IGF1 (Phyco*S*tev*IGF1). \u003c/em\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Same as in (B) upon replacement of the \u003cem\u003etev\u003c/em\u003ecleaving sequence with that of the human rhinovirus protease cleaving site (\u003cem\u003ehrv\u003c/em\u003e: LEVLFQ/GP), denoted as \u003cem\u003ecpcB*6xHis*hrv*IGF1 (Phyco*hrv*IGF1).\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e) Same as in (D) with the addition of a spacer (\u003cem\u003eS\u003c/em\u003e: PAEKWAPGGS) between the \u003cem\u003ecpcB\u003c/em\u003e and \u003cem\u003e6xHis\u003c/em\u003e, denoted as \u003cem\u003ecpcB*S*6xHis*hrv*IGF1 (Phyco*S*hrv*IGF1). \u003c/em\u003eThe location of forward and reverse primers is shown in black arrows. The nucleotide sequences of the transgenes are shown in the Supplementary Data section.\u003c/p\u003e","description":"","filename":"OnlineSlide1.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/e504a9bca7a51a93e466e5d2.png"},{"id":108350329,"identity":"6a7adc76-ac64-45e4-92c9-43ccc0a5819d","added_by":"auto","created_at":"2026-05-03 10:09:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39196,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic DNA PCR analysis testing the \u003cem\u003ecpc\u003c/em\u003e operon genotype of WT and \u003cem\u003eIGF1\u003c/em\u003e transformants. A 1-kb DNA ladder (GeneRuler, Thermoscientific) was used as molecular weight (MW) markers (MW lane). Primers US_Fwd (5ˈ-ACCTGTAGAGAAGAGTCCC-3ˈ), and DS_Rev (5ˈ-GCGGAATATTGTCAACCAG-3ˈ), as shown in Fig. 1(A-C), and primers Fwd (GTATTCACTCGGGTT), and Rev (GGTGCGGTTGATTTCATC), as shown in Fig. 1(D-E) flanking the insertion site were used to amplify the genomic DNA of the corresponding strains. (\u003cstrong\u003eA\u003c/strong\u003e) The WT strain yielded a single 1.745 kb PCR product, corresponding to the native \u003cem\u003ecpc\u003c/em\u003eoperon. The \u003cem\u003eSyn*S*tev*IFN\u003c/em\u003e and \u003cem\u003eSyn*tev*IFN\u003c/em\u003e strains yielded single 2.929 kb and 2.899 kb PCR products, respectively, corresponding to the modified \u003cem\u003ecpc\u003c/em\u003e operon DNA in these transformants (Fig. 1(A-C)). (\u003cstrong\u003eB\u003c/strong\u003e) The WT strain yielded a single 0.983 kb PCR product, corresponding to the native \u003cem\u003ecpc\u003c/em\u003eoperon, probed with shorter distance primers. The \u003cem\u003eSyn*hrv*IFN\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IFN\u003c/em\u003estrains yielded single 2.14 kb and 2.17 kb PCR products, respectively, corresponding to the modified \u003cem\u003ecpc\u003c/em\u003eoperon DNA in these transformants (Fig. 1(D-E)). Absence of wild type PCR products in the transformants is evidence of transgenic DNA copy homoplasmy.\u003c/p\u003e","description":"","filename":"OnlineSlide2.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/bc62066be61af9bbf2a8f4e0.png"},{"id":108350308,"identity":"bf2dfd33-0f99-40ac-809c-8fc48ebd3cee","added_by":"auto","created_at":"2026-05-03 10:09:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84599,"visible":true,"origin":"","legend":"\u003cp\u003ePhotoautotrophic growth curves and liquid culture coloration of \u003cem\u003eSynechocystis\u003c/em\u003e WT and \u003cem\u003eIGF1\u003c/em\u003e transformant strains in liquid culture. (\u003cstrong\u003eA\u003c/strong\u003e) Comparison of photoautotrophic growth kinetics of the WT (black circles), \u003cem\u003eSyn*S*tev*IGF1 (blue\u003c/em\u003e), \u003cem\u003eSyn*tev*IGF1 (green\u003c/em\u003e), and \u003cem\u003eΔcpc\u003c/em\u003e \u003cem\u003e(red\u003c/em\u003e) strains. Plotted is the log OD at 730 nm of the cultures, as a function of growth time. (\u003cstrong\u003eB\u003c/strong\u003e) Coloration of the cells in BG-11 liquid media from the three strains highlighting the blue-green wild type and the more greenish \u003cem\u003eSyn*tev*IGF1 \u003c/em\u003eand\u003cem\u003e Syn*S*tev*IGF1\u003c/em\u003e cultures. (\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) Effect of the presence (+A) or absence (-A) of antibiotic selectable marker (chloramphenicol 15 μg mL\u003csup\u003e-1\u003c/sup\u003e) on growth (C) and coloration (C) of transformants in liquid culture.\u003c/p\u003e","description":"","filename":"OnlineSlide3.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/77bea7d86b760228f14eea76.png"},{"id":108492869,"identity":"990a9e00-8c95-47c3-bfac-ed3c548a9a30","added_by":"auto","created_at":"2026-05-05 09:58:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63799,"visible":true,"origin":"","legend":"\u003cp\u003eMixotrphic growth curves and liquid culture coloration of \u003cem\u003eSynechocystis\u003c/em\u003e WT and \u003cem\u003eIGF1\u003c/em\u003e transformant strains in the presence (+A) or absence (-A) of antibiotic selectable marker (chloramphenicol 15 μg mL\u003csup\u003e-1\u003c/sup\u003e) in liquid culture. BG-11 media were supplemented with 5 mM glucose in this experiment. (\u003cstrong\u003eA\u003c/strong\u003e) Comparison of photoautotrophic growth kinetics of the WT and transformants. Plotted is the log OD at 730 nm of the cultures, as a function of growth time. (\u003cstrong\u003eB\u003c/strong\u003e) Coloration of the cells in BG-11 liquid media supplemented with 5 mM glucose. Presence of the latter helped to alleviate growth limitations and coloration differences seen under phototutrophic growth in the presence (+A) or absence (-A) of chloramhenicol from the growth kinetics of the transfromant strains.\u003c/p\u003e","description":"","filename":"OnlineSlide4.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/8610f401a0da96096e08319c.png"},{"id":108350332,"identity":"259b0b22-c91c-4ef1-9b6f-29d3b6d204b0","added_by":"auto","created_at":"2026-05-03 10:09:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22566,"visible":true,"origin":"","legend":"\u003cp\u003eDry celll biomass yield of wild type and IGF1 transformants under photoauthtropic (\u003cstrong\u003eA\u003c/strong\u003e) and mixotrophic (\u003cstrong\u003eB\u003c/strong\u003e) condiitons in the presence (+A) or absence (A) of 15 μg mL\u003csup\u003e-1\u003c/sup\u003e chloramphenicol selectable marker. Biomass accumulation was measured seven days after culrue inoculation, i.e., just prior to the onset of the stationay growth phase.\u003c/p\u003e","description":"","filename":"OnlineSlide5.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/62058b8cdbbd15d5f50b1449.png"},{"id":108350312,"identity":"df3a44b7-9c47-4c98-b995-ea857db0e78f","added_by":"auto","created_at":"2026-05-03 10:09:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":170209,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo absorbance spectra of intact \u003cem\u003eSynechocystis\u003c/em\u003e WT and \u003cem\u003eIGF1\u003c/em\u003e transformant strains probing for the presence of various light-harvesting pigments in the cells. All absorbance spectra were normalized to the Chl \u003cem\u003ea\u003c/em\u003e absorbance max at 680 nm (set equal to 1.0) to enable phycocyanin pigment content comparisons. (\u003cstrong\u003eA\u003c/strong\u003e) The absorbance spectra of WT, \u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e, \u003cem\u003eSyn*tev*IGF1, \u003c/em\u003eand\u003cem\u003e ∆cpc\u003c/em\u003e strains are shown in blue, purple, and green, and red colors, respectively. (\u003cstrong\u003eB\u003c/strong\u003e) The absorbance spectra of WT, \u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e, \u003cem\u003eSyn*hrv*IGF1, \u003c/em\u003eand\u003cem\u003e ∆cpc\u003c/em\u003e strains are shown in blue, purple, green, and red colors, respectively. Note the diminished phycocyanin content (A\u003csub\u003e625\u003c/sub\u003e nm) in the transformants, relative to that in the wild type.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/633e0e8ab70539c3df341363.png"},{"id":108350321,"identity":"0101820b-3dfe-45db-8ab0-12a167c06739","added_by":"auto","created_at":"2026-05-03 10:09:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":163901,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of total cell protein extracts from \u003cem\u003eSynechocystis\u003c/em\u003e WT and \u003cem\u003eIGF1\u003c/em\u003etransformant strains. (\u003cstrong\u003eA\u003c/strong\u003e) WT lanes: Coomassie-stained SDS-PAGE showing the protein profile of wild type cells. Based on Coomassie stain, the most abundant cellular proteins are the RbcL, CpcB, and CpcA. All transformants examined lacked the CpcB β-subunit band from the ~19 kDa position. Instead, they showed new protein bands in the 26-28 kDa regions. (\u003cstrong\u003eB\u003c/strong\u003e) Based on Zn-phycobilin chromophore fluorescence, protein bands migrating to 26-28 kDa were assigned to the Phyco*(S)*IGF1 fusion proteins. 1-µg Chl \u003cem\u003ea\u003c/em\u003e equivalent was loaded on the lanes for the SDS-PAGE analysis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/f0d703a47be92c0a088c3b85.png"},{"id":108350339,"identity":"8b175ba5-f2f2-4aba-b507-6c8df78bf275","added_by":"auto","created_at":"2026-05-03 10:09:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":93453,"visible":true,"origin":"","legend":"\u003cp\u003eCobalt-column differential affinity chromatography, isolation of the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 fusion protein complexes, and rTEV / rHRV protease cleaving of the IGF1 protein from the Phyco*IGF1-associated protein complex. (\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE and Coomassie-stained resolution of the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 constituent proteins. (0 IU) Shown is the presence of the 29 kDa CpcG1 linker, the 27 kDa Phyco*IGF1, and the 17 kDa CpcA α-subunit of phycocyanin. Incubation of the isolated (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 proteins with 20, 40, and 80 IU of rTEV (GenScript, Piscataway, NJ) at 30°C for 3 h showed the graduate release of the IGF1 from the heterohexameric complex, migrating to about the 7.6 kDa electrophoretic mobility position, and the concomitant appearance of a 21 kDa protein band, resulting from the de novo formation of CpcB*His*tev / Cpc*His*S*tev (marked as Phyco*) cleaving reaction products. (\u003cstrong\u003eB\u003c/strong\u003e) Zinc-chromophore fluorescence of phycocyanobilin proteins present in the samples of (A), showing loss of fluorescence intensity from the 27 kDa protein band (0 IU) and gain in this signal at the 21 kDa position, emanating from the appearance of the CpcB*His*S*tev (Phyco*) proteolysis fragment. \u0026nbsp;(\u003cstrong\u003eC\u003c/strong\u003e) Same as in (A), with the exemption of showing results with the isolated (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 proteins. Incubation of the isolated (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 proteins with 1 IU of rHRV at 4°C for 12 h was sufficient to bring the cleaving reaction to quantitative completion.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Zinc-chromophore fluorescence of phycocyanobilin proteins present in the samples of (C), showing loss of fluorescence intensity from the 27 kDa protein band (0 IU) and gain in this signal at the 21 kDa position (1 IU), emanating from the appearance of the CpcB*S*His*hrv (Phyco*) proteolysis fragment.\u0026nbsp; 20 µg of isolated fusion protein complexes s were used in the protease cleaving reactions.\u003c/p\u003e","description":"","filename":"OnlineSlide8.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/afccac02e894f0fe1b0eb11e.png"},{"id":108350317,"identity":"c75050a0-b61e-4574-8d1e-1f30507fbec7","added_by":"auto","created_at":"2026-05-03 10:09:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16359,"visible":true,"origin":"","legend":"\u003cp\u003eDensitometric analysis of protein bands from the SDS-PAGE profile shown in Figure 8. (\u003cstrong\u003eA\u003c/strong\u003e) Cleaving of the fusion protein Phyco*tev*IGF1 in the presence of rTEV at 30ºC for 3 h, and accumulation of the reaction product Phyco*tev. (\u003cstrong\u003eB\u003c/strong\u003e) Cleaving of the fusion protein Phyco*hrv*IGF1 in the presence of rHRV at 30ºC for 3 h, and accumulation of the reaction product Phyco*hrv. Amounts of Phyco*tev/hrv*IGF1 reactants, and Phyco*tev/hrv products are plotted as a function of the rTEV/rHRV concentration used. Bio-Rad Image Lab software (Hercules, CA) was used for the densitometric analysis. Results are the average of four measurements with (α,β*tev/hrv*FGF2)\u003csub\u003e3\u003c/sub\u003eG1 fusion protein samples.\u003c/p\u003e","description":"","filename":"OnlineSlide9.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/3485ee540d5f4ba32aaa453c.png"},{"id":108350319,"identity":"3adcd56f-58e4-494a-af55-12538a7f54cb","added_by":"auto","created_at":"2026-05-03 10:09:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":40497,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SDS-PAGE Coomassie stain, (\u003cstrong\u003eB\u003c/strong\u003e) Zn-chromophore labeling, and (\u003cstrong\u003eC\u003c/strong\u003e) Western blot analysis of 20 µg of isolated (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 fusion protein complexes incubated with 1 IU of rHRV protease at 30°C for 3 h. The constituent proteins were separated by SDS-PAGE and probed with IGF1-specific polyclonal antibodies. A molecular marker is presented on the gel's left side for protein molecular weight estimation.\u003c/p\u003e","description":"","filename":"OnlineSlide10.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/9342ce6eac4452f16713dda9.png"},{"id":108350333,"identity":"ec6a1e59-cd34-4bcb-94b9-dbf0778a51d7","added_by":"auto","created_at":"2026-05-03 10:09:47","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":39737,"visible":true,"origin":"","legend":"\u003cp\u003eNative PAGE and immunoblot analysis of eluted (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 and (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric complexes harboring the two different CpcB*IFN fusion constructs. Left panel shows a Coomassie stain of the respective Native-PAGE analysis proteins. Electrophoretic mobilities to about 150 kDa are consistent with the calculated molecular weights of these heterohexameric complexes. Middle panel shows an immunoblot analysis of the bands shown on the left panel with anti-CpcB antibodies. Right panel shows a immunoblot analysis with anti-IGF1 antibodies. These results showed that antigenic epitopes on CpcB and IGF1 can be inaccessible due to the conformation of the IGF1 in relation to the α,β heterohexamer.\u003c/p\u003e","description":"","filename":"OnlineSlide11.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/ccc7b191162967147116021d.png"},{"id":108350340,"identity":"8d732865-37bd-4ee1-bda4-b8d9745aa3f2","added_by":"auto","created_at":"2026-05-03 10:09:53","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":40460,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Fusion orientation of the IGF1 protein showing, by arrow, the linkage of its N-terminus to the CpcB protein C-terminus. (\u003cstrong\u003eB\u003c/strong\u003e) Fusion orientation of the IGF1 protein showing, by arrow, the linkage of its C-terminus to the CpcB protein C-terminus\u003c/p\u003e","description":"","filename":"OnlineSlide12.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/a6ea32f4ab301b5086cd9fa4.png"},{"id":108350315,"identity":"cc472b70-37b1-4be9-868c-90147f60be71","added_by":"auto","created_at":"2026-05-03 10:09:43","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":89287,"visible":true,"origin":"","legend":"\u003cp\u003eThe IGF1 fusion constructs assembled as (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 modified phycocyanin discs, containing the native CpcA α- and modified CpcB β-subunits of phycocyanin. (\u003cstrong\u003eA\u003c/strong\u003e) CpcB and IGF1 are shown in the C-to-N fusion orientation. (\u003cstrong\u003eB\u003c/strong\u003e) CpcB and IGF1 are shown in the C-to-C fusion orientation. These (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 modified discs are functionally associated with the allophycocyanin core cylinders (Hidalgo et al., 2022), thus conferring stability to the recombinant protein, while absorbing irradiance and transferring the excitation energy to the reaction center.\u0026nbsp; CpcA α-subunits, CpcB β-subunits, and IGF1 are shown in green, brown, and purple colors, respectively. The CpcG1 linker protein occupies the hollow center of the disc but is omitted from the schematic for clarity of presentation.\u003c/p\u003e","description":"","filename":"OnlineSlide13.png","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/f74d92ea3c18de3c8f77bb5a.png"},{"id":108803884,"identity":"e9b0dfc4-4795-46ba-b710-685f4b333162","added_by":"auto","created_at":"2026-05-08 15:10:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1591539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/cd7a9d13-dc88-4925-86b1-e89c4548e08e.pdf"},{"id":108350314,"identity":"d12c3886-ca00-47bf-ab35-e24d2e243b1e","added_by":"auto","created_at":"2026-05-03 10:09:43","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":232448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at https://doi\u003c/p\u003e","description":"","filename":"20260417IGF1Suppl.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9453408/v1/039fdb69196fba17d1841edd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insulin-like growth factor 1 (IGF1) overexpression on a phycocyanin carrier protein in cyanobacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInsulin-like Growth Factors (IGFs) are a class of small hormonal proteins that are primarily generated in the liver of mammals and aid in organismal cell growth and development (Maki \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Mammals, including humans and bovines, possess two different IGFs: IGF1 and IGF2 (Poreba and Durzynska \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; LeRoith et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The human and bovine \u003cem\u003eIGF1\u003c/em\u003e genes encode identical 70 amino acid long polypeptides with a molecular weight of approximately 7.6 kDa (Vajdos et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rotwein \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The tertiary configuration of the IGF1 proteins comprise an asymmetrical helix-coil protein structure, having a catalytic site closer to its N-terminus domain (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause of its importance in cell growth and development, and in addition to its use in basic research, IGF1 is in high demand as a growth supplement by the cultured meat and leather industries. This necessitates sufficient supplies to meet the growing demand. Photosynthetic organisms provide an interesting platform for recombinant IGF1 production as they do not need an external source of organic nutrients to grow (Panahi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Poudel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They primarily rely on sunlight, carbon dioxide and water. In addition, the risk of contamination is significantly lower than that of a mammalian or fermentative cell system. However, producing recombinant IGF1 in plants is time-consuming, as plants grow slowly and require complex and often unstable genetic transformation protocols. Furthermore, the stability of recombinant proteins themselves in non-native organisms, especially plants and algae, is often severely compromised by intracellular enzymes and the cellular proteasome that recognize and degrade exogenous proteins (Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), resulting in very low steady state yields (Majhi and Melis \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Such pitfalls can be alleviated using cyanobacteria.\u003c/p\u003e \u003cp\u003eUnicellular cyanobacteria offer a platform to produce a wide variety of bioactive chemicals (Lindberg et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Formighieri and Melis \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Betterle and Melis \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Price et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Santos-Merino et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Melis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and recombinant proteins (Betterle et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hidalgo Martinez et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Majhi and Melis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Melis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Majhi and Melis \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003eb\u003c/span\u003e). They grow much faster than plants and are amenable to stable genetic transformation, including spontaneous uptake of exogenous DNA and a direct double homologous recombination, leading to successful transformations. The latter enable the generation of genetically modified strains, suitable for the expression of recombinant enzymes and other proteins (Berla et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The ease of uptake of exogenous DNA, combined with homologous recombination and photoautotrophic or heterotrophic growth, makes them ideal hosts for recombinant protein production (Berla et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, stability of recombinant protein expression in cyanobacteria is enhanced by the fusion of otherwise unstable recombinant proteins with highly-expressed native proteins, e.g. phycocyanin, which has been shown to substantially improve recombinant protein stability and accumulation within the cells (Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Majhi and Melis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, the cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 (\u003cem\u003eSynechocystis\u003c/em\u003e) was used as a model organism and a host to test for the expression of IGF1. Four genetic fusion constructs between the phycocyanin CpcB β-subunit and the \u003cem\u003eIGF1\u003c/em\u003e gene were designed and successfully integrated into the \u003cem\u003eSynechocystis\u003c/em\u003e genome for recombinant IGF1 production. Two of these constructs entailed a CpcB C-terminus fusion to the IGF1 N-terminus (forward IGF1 expression). The other two entailed a CpcB C-terminus fusion to the IGF1 C-terminus (inverse IGF1 expression). The fusion constructs of forward-IGF1 and inverse-IGF1 with the β-subunit of phycocyanin were successfully expressed and accumulated in stoichiometric amounts with the CpcB protein. The synthesized Phyco*IGF1 proteins (CpcB*forward-IGF1 and CpcB*inverse-IGF1) were effectively isolated from crude cellular extracts using differential cobalt affinity column chromatography. Moreover, the IGF1 protein was successfully cleaved and separated from the Phyco*IGF1 fusion form, upon the action of recombinant proteases. The current study also examined the effect of antibiotic and glucose on \u003cem\u003eSynechocystis\u003c/em\u003e cell growth and biomass yield. It suggested a cost-effective and efficient method for using recombinant DNA technology to exploit cyanobacteria as photosynthetic cell factories in the production of IGF1.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA constructs\u003c/h2\u003e \u003cp\u003eGenetically modified cyanobacterial strains were generated upon transformation of the wild-type \u003cem\u003eSynechocystis\u003c/em\u003e. The highly expressed phycocyanin \u003cem\u003ecpc\u003c/em\u003e operon in \u003cem\u003eSynechocystis\u003c/em\u003e, consisting of \u003cem\u003ecpcB\u003c/em\u003e, \u003cem\u003ecpcA\u003c/em\u003e, \u003cem\u003ecpcC2\u003c/em\u003e, \u003cem\u003ecpcC1\u003c/em\u003e, and \u003cem\u003ecpcD\u003c/em\u003e genes that code for different subunits of the abundant phycocyanin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were the subject of this genetic transformation. The \u003cem\u003ecpc\u003c/em\u003e operon was modified upon replacing the native \u003cem\u003ecpcB\u003c/em\u003e gene, encoding the β-subunit of phycocyanin, with a fusion construct of the \u003cem\u003ecpcB\u003c/em\u003e in the leading position and the \u003cem\u003eIGF1\u003c/em\u003e in the trailing position of the linear construct. The latter was fused in the forward (N-to-C) or inverse (C-to-N) orientation (please see below). In this arrangement, the IGF1 gene was inserted prior to the stop codon at the carboxyl terminus of the \u003cem\u003ecpcB\u003c/em\u003e gene. Additional DNA sequences were inserted between the \u003cem\u003ecpcB\u003c/em\u003e and the \u003cem\u003eIGF1\u003c/em\u003e fusion, including the 6xHis tag, a spacer (S: PAEKWAPGGS), the Tobacco Etch Virus (TEV) protease cleaving sequence (tev: ENLYFQ/G), followed by the \u003cem\u003eSynechocystis\u003c/em\u003e codon optimized DNA sequence of the Insulin-Like Growth Factor 1 (IGF1). Upon double homologous recombination, the resulting fusion constructs \u003cem\u003ecpcB*6xHis*tev*IGF1\u003c/em\u003e and \u003cem\u003ecpcB*6xHis*S*tev*IGF1\u003c/em\u003e replaced the native \u003cem\u003ecpcB\u003c/em\u003e gene in the \u003cem\u003eSynechocystis\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The transgenic strains harboring the aforementioned DNA constructs in the genome were termed \u003cem\u003eSyn*tev*IGF1\u003c/em\u003e and \u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo additional fusion constructs was generated by assembling DNA sequences of \u003cem\u003ecpcB\u003c/em\u003e, a spacer (S: PAEKWAPGGS), 6xHis tag, the Human Rhinovirus 3C (HRV) protease cleaving sequence (hrv: LEVLFQ/GP), followed by the codon optimized DNA sequence of the Insulin-Like Growth Factor 1 (inv-IGF1). The resulting fusion constructs \u003cem\u003ecpcB*6xHis*hrv*IGF1\u003c/em\u003e and \u003cem\u003ecpcB*S*6xHis*hrv*IGF1\u003c/em\u003e replaced the native \u003cem\u003ecpcB\u003c/em\u003e gene in the \u003cem\u003eSynechocystis\u003c/em\u003e genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,E). The transgenic strains harboring the DNA constructs were termed \u003cem\u003eSyn*hrv*IGF1\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,E).\u003c/p\u003e \u003cp\u003eThe spacer S was designed with the inclusion of two prolines to change the relative orientation of the IGF1 protein relative to that of the CpcB β-subunit and thus to probe for potential hindrances in the assembly of the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric disc (please see discussion section). The cleaving sequences tev and hrv were introduced in the constructs to test for the cleaving efficiency of the IGF1 from the isolated (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric Phyco*IGF1 fusion protein forms. All the DNA constructs were designed in the lab and were synthesized by Biomatik USA (Wilmington, DE, United States). The resulting corresponding plasmids were used to transform wild type \u003cem\u003eSynechocystis\u003c/em\u003e, as described (Majhi and Melis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll cyanobacterial strains were maintained on BG-11 agar plates containing 1% agar, 0.3% sodium thiosulfate, 10 mM TES-NaOH (pH 8.2), and appropriate antibiotics (kanamycin, 25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or chloramphenicol, 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). All plates and cultures were kept at 25\u0026deg;C under continuous illumination at 40 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenomic DNA PCR analysis\u003c/h3\u003e\n\u003cp\u003eTo determine the genomic DNA state of transformants, colony PCR analysis was performed using cyanobacterial liquid cultures. A small aliquot of cyanobacterial culture (20 \u0026micro;L) was mixed with 100% ethanol (20 \u0026micro;L) and Chelex\u0026reg; 100 resin (100 \u0026micro;L, 10% w/v) (BioRad: 142\u0026ndash;1253), and heated at 98 \u0026ordm;C for 10 min prior to centrifugation at 16,000 g for 10 min. The supernatant was used as a DNA template for the PCR analysis, as follows. In a 25 \u0026micro;L reaction mixture, 2.5 \u0026micro;L of supernatant was combined with Q5 High-Fidelity 2X Master Mix (New England Biolab, Ipswich, MA) for the PCR reaction. The manufacturer's instructions were followed to accurately set the annealing temperature for primers \u003cem\u003eUS Fwd\u003c/em\u003e: 5ˈ-ACCTGTAGAGAAGAGTCCC-3ˈ; \u003cem\u003eDS_Rev\u003c/em\u003e: 5ˈ-GCGGAATATTGTCAACCAG-3ˈ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C), and \u003cem\u003ecpcB-Fwd\u003c/em\u003e: GTATTCACTCGGGTT; \u003cem\u003ecpcA-Rev\u003c/em\u003e: GGTGCGGTTGATTTCATC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,E),). Amplified PCR products were analyzed on a 1% agarose gel to identify the DNA PCR products and thus to test the homoplasmy status of the transformants genomic DNA.\u003c/p\u003e\n\u003ch3\u003eCell growth and dry cell weight measurement\u003c/h3\u003e\n\u003cp\u003eCells were grown in 100 mL BG-11 liquid media until OD\u003csub\u003e730\u003c/sub\u003e reached 0.8-1.0. Cells were harvested by centrifugation at 8,000 \u003cem\u003eg\u003c/em\u003e for 15 min, the supernatant was discarded, and the pellet was resuspended in 5 mL of fresh BG-11. In photoautotrophic growth cultivation, cells were inoculated to an OD\u003csub\u003e730\u003c/sub\u003e of 0.05 in 100 mL of BG-11 in the absence or presence of antibiotic, as a selectable marker. In mixotrophic growth, cells were grown in the presence of 5 mM glucose in addition to the above-mentioned conditions. Cultures were kept on a shaker (rpm: 100) at 30\u0026deg;C under continuous illumination (40 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 7 days. The OD\u003csub\u003e730\u003c/sub\u003e was measured every 24 h. The rate of growth was plotted as a function of growth time, using the GraphPad Prism or KaleidaGraph software. Following 7 days of growth, cells were pelleted by centrifugation at 8,000 \u003cem\u003eg\u003c/em\u003e for 15 min. Wet pellets were rinsed twice and dried at 90\u0026deg;C for approximately 2\u0026ndash;3 h prior to dry weight measurement. Dry cell weight data were plotted using the GraphPad Prism software.\u003c/p\u003e \u003cp\u003eWhole cell absorption spectra were measured as described (Majhi and Melis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cells were pelleted by centrifugation at 5,000 \u003cem\u003eg\u003c/em\u003e for 15 min after cultures reached an OD\u003csub\u003e730\u003c/sub\u003e of 0.8-1.0. Pellets were resuspended in aliquots of 2 mL of BG-11 prior to measurement. A Shimadzu UV-1800 spectrophotometer was used to measure the absorbance spectra, with the latter suspended at an optical cell density OD\u003csub\u003e730\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;~\u0026thinsp;0.4. The optical path length and scanning speed were set to 1 cm and 200 nm/min, respectively. GraphPad Prism or KaleidaGraph software were used to plot the resulting absorbance spectra.\u003c/p\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e concentration was measured as described in (Kirst et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Chlorophyll and carotenoid pigments were extracted from whole or broken cells using 100% methanol, as the solvent. The methanolic solution containing these pigments was used to measure the absorbance spectra. The absorbance of the methanolic extract at 663 nm was used to calculate the chlorophyll \u003cem\u003ea\u003c/em\u003e content of the samples. Similarly, the A\u003csub\u003e625\u003c/sub\u003e nm value from the whole cell absorbance spectra was used to estimate the phycocyanin content of cells.\u003c/p\u003e\n\u003ch3\u003eProtein Isolation\u003c/h3\u003e\n\u003cp\u003eCells were harvested at an OD\u003csub\u003e730\u003c/sub\u003e of 0.8-1.0 by centrifugation at 8,000 \u003cem\u003eg\u003c/em\u003e for 15 min. Pellets were resuspended in 25 mM Tris-HCl, pH-8.2, solution supplemented with cOmplete\u0026trade; mini protease inhibitor cocktail (2 mL/300 mL cell culture). The cell suspension was passed through a French Press three times at a pressure of 1,500 PSI to disrupt the cells. Unbroken cells were pelleted by centrifugation at 400 \u003cem\u003eg\u003c/em\u003e for 3 min. The 6xHis-tagged proteins were isolated from the crude cellular extracts using a differential cobalt-affinity column chromatography. Prior to the His-tagged protein isolation, the crude cellular extracts were incubated in 20 mM HEPES buffer, pH-7.5, containing 0.5% Triton-X 100 at 4\u0026deg;C for 30 min in a spinning rotor (15 cycles/min). Following incubation, the crude cellular extracts were centrifuged at 16,000 \u003cem\u003eg\u003c/em\u003e for 5 min to remove insoluble materials. The supernatant, containing soluble proteins, were mixed with cobalt-resin (HIS-Select\u0026reg; Cobalt Affinity Gel, Millipore Sigma) in a 1:1 (v:v) ratio and incubated at 4\u0026deg;C for 30\u0026ndash;45 min in a spinning rotor (15 cycles/min) to facilitate protein binding to the resin. The mixture was then passed through a column and washed four times with 20 mM HEPES, pH-7.5, 150 mM NaCl, and 10 mM imidazole washing solution (10 mL/wash) to remove unbound proteins. The His-tagged fusion proteins were eluted from the Co column using 20 mM HEPES, pH-7.5, 150 mM NaCl, and 250 mM imidazole elution solution. Eluted proteins were concentrated using Amicon\u0026reg; Ultra 15 mL centrifugal filters (Millipore Sigma). The concentrated protein samples were snap frozen in liquid nitrogen and stored at -80\u0026deg;C for later use.\u003c/p\u003e\n\u003ch3\u003eSDS-PAGE and NATIVE-PAGE analysis\u003c/h3\u003e\n\u003cp\u003eThe crude cell extracts were used to resolve the total \u003cem\u003eSynechocystis\u003c/em\u003e cellular proteins. 1 \u0026micro;g chlorophyll \u003cem\u003ea\u003c/em\u003e equivalent of crude cell extract was solubilized in 1x Laemmli Sample Buffer (Bio-Rad, Hercules, CA) for 30 min at room temperature. To facilitate solubilization and protein denaturation, 1 M urea and 5% β-mercaptoethanol were added to the mix, and samples were gently vortexed every 10 min. Following incubation, samples were centrifuged at 16,000 g for 3 min and the supernatant loaded onto a Precast 12-well kD\u0026trade; Mini-PROTEAN\u0026reg; TGX\u0026trade; Protein Gel (Bio-Rad, Hercules, CA) and electrophoresed at 200 V for 45 min. For the SDS-PAGE and NATIVE-PAGE analysis of cobalt column eluted protein samples, 20 \u0026micro;g of isolated heterohexameric phycocyanin complexes were loaded onto the gel lanes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eZinc chromophore fluorescence and Coomassie staining\u003c/h2\u003e \u003cp\u003eZinc sulphate (ZnSO\u003csub\u003e4\u003c/sub\u003e) solution was used to label the phycocyanobilin chromophore-binding proteins directly on the gel following the SDS-PAGE analysis, as follows. The electrophoresed gel was soaked in a 5 mM zinc sulphate solution for 30 min at room temperature with gentle shaking. A Chemidoc imaging system (BIORAD) was used to capture the zinc-chromophore fluorescence of the phycocyanobilin-containing protein bands, with the labeling images seen under UV light.\u003c/p\u003e \u003cp\u003eThe gel was subsequently stained in a Coomassie stain solution (0.07% brilliant blue R-250, 50% methanol, and 10% acetic acid) for 60 min at room temperature with gentle shaking to visualize and quantify specific protein bands of interest in this work. Coomassie stained gel images were taken using a Gel Doc XR+ System and Image Lab software (Bio-Rad Hercules, CA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTEV and HRV3C cleaving of the IGF1 from its carrier protein complex\u003c/h3\u003e\n\u003cp\u003eCleaving the rIGF1 from the isolated (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 Phyco*IGF1 fusion constructs was performed as follows: 20 \u0026micro;g of isolated (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 harboring the Phyco*tev*IGF1 fusion protein complexes were incubated with variable concentrations (0, 20, 40, and 80 IU) of recombinant-TEV protease (rTEV) at 30\u0026deg;C for 3 h. In case of Phyco*hrv*IGF1 protein, 20 \u0026micro;g of isolated (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric complexes harboring the Phyco*hrv*IGF1 fusion protein were incubated with 0 and 1 IU of recombinant-HRV 3C (rHRV) protease for 12\u0026ndash;16 h only, at 4\u0026deg;C. The shorter incubation in this case was chosen as a recent study (Majhi and Melis \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e) showed HRV 3C to be a more efficient protease for cleaving recombinant proteins from the CpcB*P fusion constructs. Following incubation, protein samples were prepared for SDS-PAGE analysis, as described in the SDS-PAGE subsection above. Densitometry measurements of Coomassie-stained protein bands was performed using the built-in program of the Gel Doc XR+ System (Bio-Rad, Hercules, CA).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003e20 \u0026micro;g of isolated (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric phycocyanin protein complexes were processed as described above for SDS-PAGE and NATIVE-PAGE analysis. Following the electrophoretic separation of the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 constituent proteins, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and probed with anti-CpcB and anti-IGF1 specific polyclonal antibodies as described (Majhi and Melis \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Majhi and Melis \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenomic DNA status of transformants\u003c/h2\u003e \u003cp\u003eThe genomic DNA status of transformants was evaluated to test for the attainment of homoplasmy, meaning removal of wild type copies of DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and quantitative replacement with the corresponding transgenic version harboring the heterologous fusion constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). Attaining DNA copy homoplasmy is important for determining the expression efficiency of the heterologous proteins, and the physiological and biochemical traits of genetically modified strains.\u003c/p\u003e \u003cp\u003eA colony-PCR analysis was performed with cellular DNA as a template to test the genomic DNA status of cells. The primers used in the PCR analysis were designed to cover the modified region of the \u003cem\u003ecpc\u003c/em\u003e operon in the genome, as well as the upstream and downstream native DNA regions of the transgenic DNA insertion. The PCR analysis of DNA from the wild type (WT), \u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e, and \u003cem\u003eSyn*tev*IGF1\u003c/em\u003e, showed a single product for each strain on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The product sizes were 1,745 base pairs (bp), and 2.929 bp, and 2,893 bp, respectively. Absence of a PCR product corresponding to the size of WT in the transformants is evidence that these cells achieved DNA copy homoplasmy. Similarly, transgenic strains \u003cem\u003eSyn*hrv*IGF1\u003c/em\u003e, and \u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e harboring inverse-IGF1 DNA constructs \u003cem\u003ecpcB*6xHis*hrv*IGF1\u003c/em\u003e, and \u003cem\u003ecpcB*S*6xHis*hrv*IGF1\u003c/em\u003e, respectively, exhibited a single PCR product on agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The product sizes of WT, \u003cem\u003eSyn*hrv*IGF1\u003c/em\u003e, and \u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e were 983 bp, 2,140 bp, and 2,170 bp, respectively. The results show that all of the above transgenic strains have attained a state of DNA homoplasmy, meaning they possessed only transformant DNA and lacked the original wild type copies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhotoautotrophic growth\u003c/h2\u003e \u003cp\u003eCell growth measurements are important to determine cell fitness and to assess the effect of genetic modifications on cells' ability to conduct photosynthesis and growth. The photoautotrophic growth analysis revealed that all cell types were able to use light for photosynthesis. However, the rate of growth was slower in the transformants than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The photoautotrophic growth doubling time for the Syn*tev*IGF1 and Syn*S*tev*IGF1 strains was 38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 h, which is approximately double that of the WT strain (24 h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, the transformant cultures had a more greenish coloration, compared with the WT blue green (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The slower growth and altered coloration are attributed to partial loss of phycocyanin in the transformants, as compared to that in the WT (please see below). This partial loss of the phycocyanin light-harvesting antenna is also the reason for the slower growth of the transformants under the 40 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sub-saturating intensity of irradiance employed (Kirst et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Formighieri and Melis \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The notion of a truncated phycocyanin antenna in the transformants resulting in slower rates of light absorption and slower photosynthesis and growth under these conditions is further supported by the even slower rate of growth of the Δcpc strain in which the entire cpc operon has been deleted and, thus, there is no phycocyanin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eΔcpc\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe presence (+\u0026thinsp;A) or absence (-A) of the antibiotic selectable marker (chloramphenicol 15 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the growth medium was investigated in this portion of the work. Photoautotrophic growth analysis of transgenic strains containing the inverse-IGF1 DNA constructs in the absence of antibiotic exhibited a slower rate of growth compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The doubling time for the WT was 24 h, whereas the doubling times of the \u003cem\u003eSyn*hrv*IGF1\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e were closer to 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1 h for the Syn*hrv*IGF1-A and Syn*S*hrv*IGF1-A strains. Interestingly, the presence of the selectable marked antibiotic (chloramphenicol) in the growth medium of the transformants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cem\u003eSyn*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e) caused a further slowdown in growth to 37.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Slower photoautotrophic growth in the presence of the antibiotic chloramphenicol may be linked to the extra metabolic load for the synthesis and accumulation of the chloramphenicol resistance cassette in the cells, and possibly due to adverse reactive oxygen species (ROS) production that cause damage to the photosynthetic apparatus. Chloramphenicol has been reported to promote ROS production under illumination conditions (Kodru et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this respect, cells growing in the presence of antibiotic appeared to have a lighter green coloration, compared to cells grown without antibiotic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), consistent with the above-mentioned growth mitigating conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMixotrophic growth\u003c/h2\u003e \u003cp\u003eCell growth was also measured in the presence of 5 mM glucose, with or without antibiotic, to test for the effect of an external carbon source on cell doubling time. In this case, the presence glucose enhanced the rate of growth of all strains and mitigated the effect of antibiotic that was observed under photoautotrophic conditions. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The doubling time for WT was lowered to 17 h, that of the \u003cem\u003eSyn*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e strains to 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 h, whereas in the absence of antibiotic \u003cem\u003eSyn*hrv*IGF1-A\u003c/em\u003e and \u003cem\u003eSyn*S*hrv*IGF1-A\u003c/em\u003e strains exhibited a faster growth of 18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 h. Faster growth in the presence 5 mM glucose is linked to the supplementary cellular activities, fueled in this case by both photosynthesis and glucose metabolism. Consistent with this notion is the coloration of the cultures, which was not adversely affected in the glucose-assisted cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiomass yield\u003c/h2\u003e \u003cp\u003eBiomass accumulation is also an important variable in synthetic biology as it is linked to constitutive bioproducts yield. In this study, experiments were conducted to measure the dry cell weight (dcw) of all strains employed, including WT and transgenics harboring the IGF1 protein in different fusion construct configurations. Under photoautotrophic growth conditions, WT cells accumulated approximately 0.66 g/L dcw (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e compared to 0.78 g/L dcw under mixotrophic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, under photoautotrophic cultivation conditions, transgenic strains \u003cem\u003eSyn*hrv*IGF1-A, Syn*S*hrv*IGF1-A, Syn*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e, and \u003cem\u003eSyn*S*hrv*IGF1\u0026thinsp;+\u0026thinsp;A\u003c/em\u003e, accumulated 0.58 g/L, 0.55 g/L, 0.38 g/L, and 0.32 g dcw per L culture, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Interestingly, under mixotrophic cultivation conditions such differences in were largely alleviated, resulting in greater biomass yield, on the average in the range of 0.775\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 g dcw per L in all five strains examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhycocyanin content\u003c/h2\u003e \u003cp\u003eTo determine the relative amount of phycocyanin assembled in cells, absorbance spectra of whole cell suspensions were measured. The absorbance spectrum of WT cells revealed four distinct bands with peaks at 440, 490, 625, and 680 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,C). The absorbance peak at 440 nm is attributed to chlorophyll \u003cem\u003ea\u003c/em\u003e, which is associated with photosystems I and II, whereas the chlorophyll-overlapping band at 490 nm is attributed to cellular carotenoids. The 625 and 680 nm bands (shown in greater resolution in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,D) are attributed to phycocyanin and chlorophyll \u003cem\u003ea\u003c/em\u003e, respectively. The absorbance spectrum on the phycocyanin-lacking ∆\u003cem\u003ecpc\u003c/em\u003e strain, in which the entire \u003cem\u003ecpc\u003c/em\u003e operon has been deleted (Kirst et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), is also shown. In the ∆\u003cem\u003ecpc\u003c/em\u003e strain, dominant are the chlorophyll \u003cem\u003ea\u003c/em\u003e and carotenoid pigments, with the former showing a distinct 680 nm band and a lower amplitude chlorophyll \u003cem\u003ea\u003c/em\u003e satellite peak at 625 nm. All four absorbance spectra have been normalized to the absorbance maximum of chlorophyll \u003cem\u003ea\u003c/em\u003e at 680 nm to enable comparison in the cellular composition of phycocyanin among the four strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absorbance spectra of transformants \u003cem\u003eSyn*tev*IGF1\u003c/em\u003e, \u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e and \u003cem\u003eSyn*hrv*IGF1, Syn*S*hrv*IGF1\u003c/em\u003e exhibited a lower amplitude peak at 625 nm, corresponding to phycocyanin, compared to that of WT, showing a lower phycocyanin content in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). With the A\u003csub\u003e625\u003c/sub\u003e of the ∆\u003cem\u003ecpc\u003c/em\u003e strain as baseline, we calculated the phycocyanin content in the \u003cem\u003eSyn*tev*IGF1\u003c/em\u003e, \u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e transformants to be 20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0% of that in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similarly, the phycocyanin content in the \u003cem\u003eSyn*hrv*IGF1, Syn*S*hrv*IGF1\u003c/em\u003e strains was estimated to be equal to 17.9% \u0026plusmn; 2.0% of that in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results are consistent with similar findings by Hidalgo Martinez et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, suggesting the assembly of only the proximal to the core disc of phycocyanin, with a modified the CpcB*IGF1 β-subunit, and absence of the middle and distant phycocyanin rod discs. Thus, the modified CpcB*IGF1 β-subunit with the attendant fusion construct must have caused the assembly of a truncated phycocyanin rod.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTotal cell protein analysis and fusion protein isolation\u003c/h2\u003e \u003cp\u003eTotal cellular protein analysis was performed to test for the presence of Phyco*IGF1 fusion proteins in the various transformants. Crude cellular extracts were analyzed through SDS-PAGE Coomassie stain and zinc-phycobilin chromophore fluorescence measurements. Coomassie stain results for WT exhibited three main protein bands migrating to 55, 19, and 17 kDa corresponding to the large subunit of Rubisco (RbcL), β-subunit (CpcB), and α-subunit (CpcA) of phycocyanin, alongside other cellular proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In all transgenic strains (\u003cem\u003eSyn*tev*IGF1, Syn*S*tev*IGF1, Syn*hrv*IGF1\u003c/em\u003e, and \u003cem\u003eSyn*S*hrv*IGF1)\u003c/em\u003e, protein bands corresponding to RbcL and CpcA were still observed. However, the amount CpcA was lower in the transgenics compared to WT. Furthermore, all transgenic strains lacked the 19 kDa CpcB protein band. Instead, a newly formed protein band migrating to ~\u0026thinsp;26\u0026ndash;28 kDa was observed. The latter originated from the CpcB*IGF1 fusion proteins, a conclusion supported by the pronounced zinc-phycobilin fluorescence labeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, a 23 kDa protein band was observed in the protein extracts of transgenic strains, corresponding to the chloramphenicol antibiotic cassette (CmR), which was introduced as a selection marker.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric complexes and cleaving of IGF1 from the CpcB*IGF1 fusion\u003c/h2\u003e \u003cp\u003eTo cleave the natural form of the IGF1 from the fusion proteins, recombinant constructs were designed to include a cleaving sequence (\u003cem\u003etev\u003c/em\u003e: ENLYFQ/G) specific for the Tobacco Etch Virus (TEV) protease, or (\u003cem\u003ehrv\u003c/em\u003e: LEVLFQ/GP) human rhinovirus 3C protease, placed immediately upstream from the \u003cem\u003eIGF1\u003c/em\u003e DNA sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). His-tagged fusion proteins were isolated through differential cobalt-affinity column chromatography from the crude cellular extracts as heterohexameric phycocyanin (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 discs (Hidalgo Martinez et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), in which α is the α-subunit of phycocyanin, β*IGF1 is the CpcB*His*(S)*tev*IGF1, or CpcB*(S)*His*hrv*IGF1 fusion protein, and CpcG1 is the proximal phycocyanin disc linker that anchors this phycocyanin disc to the allophycocyanin core cylinders (Hidalgo Martinez et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The isolated heterohexameric complexes served as templates for the IGF1 cleaving reactions. In the case of the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1, the cleaving reaction mixture involved incubating a fixed amount of isolated (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 protein complexes (20 \u0026micro;g protein) with different concentrations (0\u0026ndash;80 IU) of commercially available recombinant rTEV protease at 30\u0026deg;C for 3 h. Following incubation, reaction mixture samples were analyzed by SDS-PAGE to determine the cleaving and separation efficiency of the IGF1 protein from the fusion protein complexes.\u003c/p\u003e \u003cp\u003eDifferential Co-affinity chromatography isolates and their analysis by SDS-PAGE from the Syn*S*tev*IGF1 strain showed three main protein bands migrating to 29, 27, and 17 kDa, corresponding to the linker CpcG1, Phyco*IGF1, and CpcA polypeptides, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, 0 \u003cb\u003eIU\u003c/b\u003e). Addition of rTEV (20, 40, and 80 IU) to the reaction mixture harboring the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 complex resulted in the progressively greater appearance of cleaved Phyco* (20.7 kDa) and free IGF1 (~\u0026thinsp;10 kDa) protein bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B). Protease cleaving efficiency was determined from the progressive decrease in the amount of the 27 kDa Phyco*IGF1 fusion protein and the concomitant appearance of the cleaved\u0026thinsp;~\u0026thinsp;20.7 kDa Phyco* moiety that was observed as a function of the increasing amount of rTEV employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Densitometry analysis of Coomassie-stained protein gels showed that the fusion protein Phyco*IGF1 decreased to 40%, 38%, and 25% of the original in the presence of 20, 40, and 80 IU of rTEV protease, respectively. Correspondingly, the amount of the cleaved Phyco* product was measured to be 60%, 70%, and 75% of the original Phyco*IGF1 in the presence of 20, 40, and 80 IU of rTEV protease, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, when (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric phycocyanin complexes harboring the hrv cleaving sequence were incubated in the presence of 0 IU and 1 IU of HRV protease at 4\u0026deg;C for 16 h, a greater cleaving efficiency was observed on Coomassie stained SDS-PAGE. Protein samples without HRV-treatment showed three main protein bands migrating to 29, 27 and 17 kDa corresponding to CpcG1, Phyco*IGF1, and CpcA proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). The HRV-treated sample showed, in addition to Phyco*IGF1 (27 kDa) and CpcA (17 kDa), two new protein bands migrating to 24 kDa, originating from the added recombinant HRV, and ~\u0026thinsp;21 kDa, originating from the cleaved Phyco*IGF1. Densitometry analysis of protein bands from the Coomassie stained SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), showed that incubation of the isolated (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric phycocyanin complexes, harboring the hrv cleaving sequence, with 1 IU of HRV protease caused a decrease of Phyco*hrv*IGF1 proteins to about 12% of the original. Conversely, the cleaved Phyco* product accounted for about 88% of the original (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eA side-by-side analysis of SDS-PAGE Coomassie stain, Zn-phycobilin chromophore fluorescence, and Westen blot analysis was undertaken to probe for the presence and location of Phyco*IGF1 and the cleaved IGF1 in the protease treated protein samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The combined analysis revolved around the HRV reaction mixture proteins Phyco*IGF1 (~\u0026thinsp;27 kDa), rHRV (~\u0026thinsp;24 kDa), Phyco* (~\u0026thinsp;21 kDa), CpcA (~\u0026thinsp;17 kDa), and IGF1 (~\u0026thinsp;7.6 kDa), all of which were detected in the Coomassie stain (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). Three of these protein bands (Phyco*IGF1, Phyco*, and CpcA were detected with the Zn-phycobilin chromophore fluorescence method (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB), and only two (Phyco*IGF1 and IGF1) cross reacted in the Western blot analysis, conducted with specific anti-IGF1 polyclonal antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDifferential antibody access to the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 and (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 complexes\u003c/h2\u003e \u003cp\u003eSpecific polyclonal antibodies raised against the CpcB (anti-CpcB) and IGF1 (anti-IGF1) were employed to probe the stereochemistry and peripheral surface properties of the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 and (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 complexes. Native-PAGE and immunoblot analysis results of the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 and (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric complexes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. These undissociated complexes migrated to about 150 kDa, consistent with their protein composition. The immunoblot analysis showed that both anti-CpcB and anti-IGF1 strongly cross-reacted with the (α,β*tev*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 complex. Surprisingly, the same antibodies failed to cross-react with the (α,β*hrv*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 complex, suggesting a substantially different stereochemistry at the periphery of this complex, one that prevented the antibodies from accessing the antigenic epitopes of either the CpcB or IGF1 proteins. This unexpected result is rationalized upon consideration of the cleaving amino acid sequence of tev versus hrv, and in view of the inverse orientation on the IGF1 in relation to the CpcB protein. More specifically, the hrv cleaving sequence contains a proline amino acid, whereas the tev sequence does not. The proline is expected to change the angle of the following IGF1 in the fusion construct, leading to a substantially different orientation in relation to the carrier heterohexamer, whereas this did not happen with the tev sequence. In addition to the proline in the hrv cleaving sequence, there is another important difference between the two fusion construct samples examined in the Native-PAGE and immunoblot of Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. Namely, by design, there is a C-to-N forward configuration between CpcB and IGF in the (α,β*tev*IGF1)3G1 construct, but a C-to-C inverse configuration for the IGF1 in the (α,β*hrv*IGF1)3G1 construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). It is concluded that a proline in the hrv and inverse C-to-C fusion of the IGF1 in relation to the CpcB protein in the α,β heterohexamer contributed to structural properties that underline the differential immunoblot results in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e (please see discussion below).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStructural aspects of phycocyanin\u003c/h2\u003e \u003cp\u003ePhycobilisomes are the major pigment-protein complexes found in cyanobacteria, and they are an important component of the photosynthetic light harvesting system in these microorganisms (Singh et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). They comprise large, multimeric, water-soluble protein complexes peripherally associated with the cytoplasmic side of the thylakoid membrane, where they are bound to the transmembrane CP43/CP47 chlorophyll-proteins of photosystem II (Kirst et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dom\u0026iacute;nguez-Mart\u0026iacute;n et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Functionally, the phycobilisome plays an important role in the light-reactions of photosynthesis, capturing light energy and transferring the excitation energy to photosystem II, thereby enhancing water (H\u003csub\u003e2\u003c/sub\u003eO) oxidation and electron transport in the thylakoid membrane.\u003c/p\u003e \u003cp\u003ePhycobilisomes in \u003cem\u003eSynechocystis\u003c/em\u003e possess three allophycocyanin core cylinders, and six phycocyanin-containing peripheral rods (Kirst et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dom\u0026iacute;nguez-Mart\u0026iacute;n et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Each of the peripheral rods comprises 6 heterohexameric (α,β)₃ discs, consisting of three α-CpcA and three β-CpcB subunits stacked to form the phycocyanin rods and stabilized through their association with colorless linker polypeptides (Singh et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The peripheral rods are linked to the core cylinders via the colorless CpcG1 linker polypeptide, which is required for the structural and functional association of phycocyanin to allophycocyanin (Dom\u0026iacute;nguez-Mart\u0026iacute;n et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidalgo Martinez et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The CpcB and CpcA subunits of phycocyanin are the most abundant proteins in cyanobacteria (Betterle et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hidalgo Martinez and Melis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eIGF1 protein and the CpcB*IGF1 fusion folding models\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eIGF1\u003c/em\u003e gene encodes a protein of 70 amino acids, which has a folded helix-loop secondary structure (Nickle et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The mature IGF1 protein shows a non-folding short N-terminal sequence, three core α-helices and their connecting loops, and an extended C-terminal loop (Ornitz and Itoh \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). In mammal and human physiology, the IGF1 protein binds onto the receptor IGF1R protein through multiple interacting residues, which are distributed on the N-terminal, core helix, and C-terminus of IGF1 (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that both N- and C-terminal residues of IGF1 are important for proper receptor binding and eliciting the growth signal. With this in mind, we synthesized two different fusion-constructs resulting in the stable and properly folded IGF1 protein, with receptor binding residues exposed at the C-terminal or N-terminal regions of the fusion protein. Fusion construct (Phyco*tev*IGF1) was designed to link the CpcB C-terminus to the IGF1 N- terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA), thereby exposing the IGF1 C-terminus to the medium in the periphery of the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric phycocyanin disc. Phyco*hrv*IGF1 was designed to link the CpcB C-terminus to the IGF1 C- terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB), thereby exposing the IGF1 N-terminus to the medium in the periphery of the (α,β*IGF1)\u003csub\u003e3\u003c/sub\u003eG1 heterohexameric phycocyanin disc.\u003c/p\u003e \u003cp\u003eProtein folding models showed that, in strains harboring the Phyco*IGF1 (C-to-N fusion; Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA) or Phyco*IGF1 (C-to-C fusion, Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB) constructs, the IGF1 protein is localized peripherally and away from the center of the assembled (α,β)₃ discs, supported by the functional association of the (α,β*IGF1)₃G1 discs with the allophycocyanin core cylinders, and from the stability of IGF1 in these transformants (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This configuration allowed the rTEV and rHRV proteases to readily access the cleaving tev (ENLYFQ/G) and hrv (LEVLFQ/GP) sequences, respectively, resulting in the release of the IGF1 protein in the reaction medium upon its separation from the Phyco*IGF1 fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Additional support for the cytosolic exposure of the fusion construct to the medium is derived from the functional association of the His-tag with cobalt in the differential cobalt (α,β*IGF1)₃G1 disc association and release upon treatment with imidazole.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, structural and functional differences existed between the (α,β*tev*IGF1)₃G1 and (α,β*hrv*IGF1)₃G1 complexes, specifically as these pertain to anti-CpcB and anti-IGF1 antibody recognition of the pertinent epitopes in the (α,β*hrv*IGF1)₃G1 complex only (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). These results suggest that caution should be exercised, when fusion constructs are designed, to take into consideration the alignment of the target protein in relation to the (α,β) heterohexameric career disc.\u003c/p\u003e \u003cp\u003eThe above are important considerations in synthetic biology, where high amounts of recombinant proteins properly folded for use in the biopharmaceutical sector, or catalytically active enzymes are required for enhanced pathway yield toward the synthesis of bioactive compounds and proteins. The plant and human proteins referenced in work from this lab (Melis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) would not accumulate in heterologous systems, e.g., cyanobacteria, when expressed by themselves, albeit under the control of a strong promoter (Formighieri and Melis \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Majhi and Melis \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e), evidently due to the cellular proteasome activity designed to degrade denatured and foreign proteins (Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This recombinant protein stability problem is apparently alleviated by the fusion constructs technology.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eChl: Chlorophyll\u003c/p\u003e\n\u003cp\u003eCar: Carotenoids\u003c/p\u003e\n\u003cp\u003ePhc: Phycocyanin\u003c/p\u003e\n\u003cp\u003eApc: Allophycocyanin\u003c/p\u003e\n\u003cp\u003ePBS: phycobilisome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ecpc\u003c/em\u003e\u003c/strong\u003e: The phycocyanin-encoding operon in cyanobacteria.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecpcA\u003c/em\u003e: Gene encoding the phycocyanin \u0026alpha;-subunit (sll1578)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecpcB\u003c/em\u003e: Gene encoding the phycocyanin \u0026beta;-subunit (sll1577)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ecpcG1\u003c/em\u003e: Gene encoding the phycocyanin-to-allophycocyanin proximal linker protein CpcG1 (slr2051)\u003c/p\u003e\n\u003cp\u003e(\u0026alpha;,\u0026beta;)\u003csub\u003e3\u003c/sub\u003eG1: Native heterohexameric phycocyanin disc with the proximal phycocyanin-to-allophycocyanin linker protein CpcG1\u003c/p\u003e\n\u003cp\u003eIGF1: Insulin like growth factor-1\u003c/p\u003e\n\u003cp\u003e(\u0026alpha;,\u0026beta;*IGF1)\u003csub\u003e3\u003c/sub\u003eG1: Heterohexameric phycocyanin disc with a fusion construct of the CpcB \u0026beta;-subunit in the leading and the IGF1 in the trailing sequence position\u003c/p\u003e\n\u003cp\u003eRbcL: The large subunit of Rubisco\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynechocystis: Synechocystis\u003c/em\u003e sp. PCC 6803\u003c/p\u003e\n\u003cp\u003eS: Oligopeptide spacer (PAEKWAPGGS)\u003c/p\u003e\n\u003cp\u003erTEV: Recombinant \u003cu\u003eT\u003c/u\u003eobacco \u003cu\u003eE\u003c/u\u003etch \u003cu\u003eV\u003c/u\u003eirus protease\u003c/p\u003e\n\u003cp\u003etev: Tobacco Etch Virus amino acid cleaving sequence (ENLYFQ/G)\u003c/p\u003e\n\u003cp\u003erHRV: Recombinant Human Rhinovirus 3C proteases\u003c/p\u003e\n\u003cp\u003ehrv: Human Rhinovirus amino acid cleaving sequence (LEVLFQ/GP)\u003c/p\u003e\n\u003cp\u003ePhyco*tev*IGF1: the CpcB*His*(S)*tev*IGF1 construct protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhyco*hrv*IGF1: the CpcB*(S)*His*hrv*IGF1 construct protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSyn*tev*IGF1\u003c/em\u003e: Transformant strain harboring the \u003cem\u003ecpcB*6xHis*tev*IGF1\u003c/em\u003e construct.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSyn*S*tev*IGF1\u003c/em\u003e: Transformant strain harboring the \u003cem\u003ecpcB*6xHis*S*tev*IGF1\u003c/em\u003e construct.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSyn*hrv*IGF1\u003c/em\u003e: Transformant strain harboring the \u003cem\u003ecpcB*6xHis*hrv*IGF1\u003c/em\u003e construct.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSyn*S*hrv*IGF1\u003c/em\u003e: Transformant strain harboring the \u003cem\u003ecpcB*S*6xHis*hrv*IGF1\u003c/em\u003e construct.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWT: wild type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBharat Kumar Majhi (BKM) and Anastasios Melis (AM) designed the project. BKM conducted the experimental work. AM quantified some of the results. BKM drafted the figures and wrote the first version of the article, which was edited by AM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no competing financial interests that could have influenced the work reported in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by University of California Fund # 63742.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this work are included in this published version and the associated supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerla BM, Saha R, Immethun CM, Maranas CD, Moon TS, Pakrasi HB (2013) Synthetic biology of cyanobacteria: unique challenges and opportunities. 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Nature Communications 12 (1):5497. doi: https://doi.org/10.1038/s41467-021-25813-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fusion DNA and protein constructs, Phycocyanin, Recombinant protein stability, Synechocystis sp. PCC 6803","lastPublishedDoi":"10.21203/rs.3.rs-9453408/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9453408/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Insulin-like Growth Factor 1 (IGF1) is a small (~\u0026thinsp;7.6 kDa) protein-hormone primarily generated in the liver of mammals. It has a similar structure to insulin, and functions to support cell growth and development. As a result, IGF1 is in high demand in the biopharmaceutical, biomedical, and research fields. In eukaryotic photosynthetic systems, expression of this and other mammal proteins has been met with limited success. The current study used \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803, a freshwater, single-celled cyanobacterium, as a host system to produce the IGF1 through recombinant DNA technology. To stabilize and enhance IGF1 accumulation in the cells, fusion constructs of the codon optimized \u003cem\u003eIGF1\u003c/em\u003e gene with the β-subunit (\u003cem\u003ecpcB\u003c/em\u003e gene) of phycocyanin were designed. The resulting transgenic strains accumulated significant amounts of recombinant IGF1 in direct proportion to phycocyanin in \u003cem\u003eSynechocystis\u003c/em\u003e. Quantitative aspects of the work, including the culture biomass yield in photoautotrophic and mixotrophic growth conditions, and the amount of recombinant fusion protein (CpcB*IGF1), as a fraction of total cellular protein in \u003cem\u003eSynechocystis\u003c/em\u003e are reported. Further, antibody specificity is used as a tool to examine the effect of different fusion orientations between the CpcB and IGF1 proteins, needed to inform of optimal conditions for IGF1 functional properties. The work adds to the concept of cyanobacteria serving as cell factories for the renewable photosynthetic generation of biopharmaceutical proteins from sunlight, carbon dioxide and water.\u003c/p\u003e","manuscriptTitle":"Insulin-like growth factor 1 (IGF1) overexpression on a phycocyanin carrier protein in cyanobacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-03 10:09:05","doi":"10.21203/rs.3.rs-9453408/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-22T13:29:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59957107932904466902906240431269982299","date":"2026-04-21T07:34:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T06:25:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T06:04:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T18:25:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Phycology","date":"2026-04-18T01:40:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"journal-of-applied-phycology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10811","submissionUrl":"https://submission.nature.com/new-submission/10811/3","title":"Journal of Applied Phycology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"44af53f0-fb7d-421d-917a-86adb74979b7","owner":[],"postedDate":"May 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-03T10:09:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-03 10:09:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9453408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9453408","identity":"rs-9453408","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}