Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme

Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme | 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 Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme Hamid Shahbazmohammadi, Eskandar Omidinia This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7604522/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Streptokinase (SK, EC 3.4.99.0) is an enzyme produced by beta-haemolytic streptococcus , and catalyzes the conversion of plasminogen to its active and proteolytic form, plasmin. SK is used in medicine as a fibrinolytic enzyme system for dissolving clots in conditions such as heart attacks, ling artery emboli, vein thrombosis, and occlusions of arteries. Despite the impressive use of this thrombolytic drug, its immunogenicity and short biological half-life are major challenges that limit its efficacy in clinical setting. In this communication, we studied the polysialylation of SK with the aim to improve the pharmacokinetics of this drug in medicine. The skc-2 gene from S. equisimilis ATCC 9542 was codon optimized, and produced as a recombinant enzyme in Escherichia coli W3110. Recombinant SK was covalently conjugated to oxidized polysialic acid (PSA; also referred to as colominic acid; CA) via reductive amination in the presence of NaCNBH 3 . The native and polysialylated variants were compared in terms of structural properties, enzyme kinetics, stability, immunization and biological half-life. The best molecular weight of PSA, optimum molar ratio, incubation time, and temperature for conjugation reaction of PSA to SK were determined to be 10.0 kDa, 200: 1, 24 h and 25°C, respectively. SDS-PAGE analysis revealed a band at 55.0 kDa for conjugated SK which confirmed the conjugation of PSA to SK. The exact molecular weight of SK-10.0 kDa PSA was determined to be 56.5 kDa by MALDI-TOF spectrometry mass which matches the calculated value by SDS-PAGE. Polysialylation induced a decrease in the far UV CD signal, suggesting an increase in the protein alpha helix content. The intrinsic fluorescence intensity of polysialylated SK increased compared to the native version, meaning that stability of SK was increased by immobilizing on PSA polymer, consistent with the CD results. K m of polysialylated SK was slightly higher than that of native SK, which showed that the attached PSA molecules to the enzyme did not significantly reduce the substrate specificity. Polysialylated SK elicited nearly 63.0% lower antibody production compared to the native variant. Native and polysialylated SKs exhibited plasma half-life of 0.5 and 2.21 h, respectively, implying that the modified variant has a 4.42-fold longer residence time in body. Briefly, comparative studies with native and PSA-conjugated enzymes show that polysialylation can be useful in enhancing the therapeutic efficacy of SK. It is worth emphasizing that this is the first report describing the use of polysialylation technology to improve the pharmaceutical properties of SK. Conjugation Half-life Immunogenicity Polysialic acid (PSA) Streptokinase (SK) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Streptokinase (SK, EC 3.4.99.0) is an enzyme produced by various strains of beta-hemolytic streptococci, and used as a thrombolytic drug for the treatment of acute myocardial infarction and pulmonary embolism. 1 – 3 SK is a member of a group of medications called as fibrinolytics, and works by catalyzing the conversion of plasminogen to the active enzyme plasmin. It forms a 1:1 stoichiometric complex with plasminogen in plasma, and produces a fibrinolytic activator. 4 , 5 This complex hydrolyzes plasminogen molecules to generate plasmin, which subsequently degrades fibrin clots, and lyses thrombi and emboli. 6 – 8 SK is in widespread clinical practice to treat acute infarction because of its function as an activator of vascular fibrinolysis. 9 – 11 However, an important clinical problem of enzyme therapy with SK is its immunogenicity. The SK enzyme is a bacterial product, and its administration develops elevated antibody titers that cause serious allergic reactions, and neutralize drug activity. As a result, this drug should not be reused within four days of the initial dose to avoid reduced effectiveness and potential allergic reactions. 12 , 13 Further thrombotic events can be treated with urokinase and tissue plasminogen activator (t-PA), which are not endangered by allergic reaction and anaphylaxis. However, they are expensive, and are not accessible to broader public. 14 , 15 Thus, SK continues to be the primary treatment for thrombolytic therapy because of its cost-efficiency. 16 Given the clinical importance of this enzyme, various attempts have been done to modify SK to increase its half-life, minimize immunogenicity, and enhance its effectiveness in the body. These studies include selective removing or replacing of some amino acids, immobilization in insoluble carrier, encapsulation in nanoparticles and PEGylation. 13 , 17 , 18 For example, removing 42 amino acids from C-terminal or replacing Lys59 residue with glutamic acid in SK have been reported to reduce immune response, and improve biological activity. 19 PEGylation has been tried as a method to prolong the half-life, and decrease the immunogenic response of SK. However, this alteration hindered its effectiveness in activating plasminogen. 14 , 18 Hasanpour and coworkers (2021) encapsulated SK in mPEG-PLGA nanoparticles to improve its pharmacokinetic properties which resulted in prolonged half-life up to 120 minutes. 13 In 2020, Baharifar et al. prepared PEG-grafted chitosan/SK nanoparticles, and increased biological circulation time of SK up to 120 minutes. 12 In a research by Sawhney et al. , a truncated PEGylated SK was designed and produced. They reported higher in vivo half-life, improved clot-specificity and reduced immune-reactivity compared to the native SK. 14 In another work, Sawhney and other colleagues employed site-specific PEGylation, and extended circulating in vivo half-life up to 20-fold compared to that of native unconjugated SK. 18 However, up to now, no PEGylated SK has received approval of Food and Drug Administration (FDA). In some cases, the development of antibodies against PEGylated medications has been documented, posing a significant drawback for the use of PEG. 20 – 22 So, there is a necessity to discover natural polymers that can decrease the immunogenicity of SK while maintaining its catalytic function. Polysialic acid (PSA), a polymer of N-acetyl neuraminic acid, is one example of natural and biodegradable polysaccharides, which can be utilized for the conjugation of bio-therapeutics. Polysialylation technology with the use of PSA (namely colominic acid; CA) as a substitute for PEG in reducing immunogensity, and extending the circulatory half-lives of proteins has been proposed. 23 . 24 The rationale of this approach is that PSA creates a liquid cloud surrounding the healing molecule, and mask the immunogenic determinants, and hence increase the circulatory half-life. 25 In the process of conjugation, activated PSA react with lysine or N-terminus of the protein to form a stable linkage. 26 , 27 Polysialylated proteins exhibited enhanced stability and functionality, increased pharmacological effectiveness, reduced immunogenicity, and decreased antigenicity. The successful application of polysialylation for other bio-molecules, e.g., insulin, 27 catalase, 28 asparaginase, 29,30 epirubicin, 25,31 and uricase 32 has also been reported. This work aimed to design and develop a polysialylated variant of SK for drug delivery with reduced immunogenicity and improved half-life. For this purpose, for the first time, we chemically synthesized a bioconjugate of recombinant SK with PSA, and examined its biochemical, structural, immunological, and pharmacokinetic characteristics as compared to the native SK. 2. Experimental Procedures 2.1. Materials CA (sodium salt) from Escherichia coli K1 (average molecular weight (MW) 10 kDa) was procured from Fluka. CA with average MW 30.0 kDa was prepared from BIOSYNTH Ltd (United Kingdom). Sodium cyanoborohydride (NaCNBH3), o -phenylenediamine (OPD), L-tryptophan (L-Trp) and 3-indoleacrylic acid (IAA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-labelled anti-rat antibodies against rat IgG and IgM were purchased from Sera-lab (Crawley Down, Sussex, UK). H-o-Val-Leu-Lys-p-nitroanilide (S-2251) was procured from Chromogenix (Italy).The pEKG-3 vector was achieved as a gift from the production department of Pasteur Institute of Iran, Karaj, Iran. Sephadex G-100 was obtained from Amersham International (Amersham, Buckinghamshire, UK). All remaining chemicals were of analytical quality. 2.2. Cloning, expression and purification of recombinant SK The cDNA sequence of S. equisimilis H46A SK (accession number: X61766) was initially retrieved from Refseq of NCBI. The nucleotide sequence that encodes SK (1248 bp) was optimized for high expression in E. coli strain W3110 using the Optimizer program from http://genomes.urv.es/OPTIMIZER , and produced by Biomatik Company (Canada). The optimized synthetic SK gene was amplified by polymerase chain reaction (PCR) with specific primers SKFw (5'-GA GAATTC ATGATTGCTGGACCT-3') and SKRev (5'-AC GGATCC TTATTTGTCGTTAGG-3'), which contained the restriction sites for Eco RI and Bam HI, respectively. The introduced restriction sites Eco RI (forward) and Bam HI (reverse) are underlined. For sequencing pEKG-3 plasmid, a forward sequencing primer (5'-TTGACAATTAATCATCGAAC-3') designed based on the Trp promoter region was used. The PCR product was cut with Eco RI and Bam HI restriction enzymes, gel purified and then ligated into the pEKG-3 expression vector previously digested with the same enzymes. The resulting recombinant construct was named pEKG-SK. The expression vector was transformed into competent cell E. coli W3110, and cultured in 30 mL M9 medium containing 150 µg/mL L-Trp and 100 µ g/mL ampicillin under agitation at 37 ºC and 180 rpm for an overnight. This overnight culture was diluted 1:10 with 300 mL M9 medium containing ampicillin, but lacking L-Trp, which was grown at 37 ºC and 180 rpm for 1.5 h. Then, trpE promoter of recombinant gene was induced under optimized conditions by adding 20 µg/mL of IAA at 37 ºC and 180 rpm for 3 h. The cells underwent centrifugation to be gathered, then rinsed with 0.9% normal saline, resuspended in lysis buffer (consisting of 50 mM NaH 2 PO 4 , 300 mM NaCl, and pH = 8.0), and were sonicated on ice for 12 minutes with a 15s on and 10s off cycle with a power frequency of 9.0 kHz. The inclusion bodies (IBs) containing recombinant SK enzyme was precipitated by centrifugation (12,000 rpm for 30 minutes at 4°C), and washed twice with wash buffer (50 mM NaH 2 PO 4 , 50 mM NaCl, 10 mM EDTA, 1.0% Triton X-100 and pH 8.0,). The washed pellet was resuspended in solublization buffer (50 mM Tris–HCl, 100 mM NaCl, 10 mM EDTA, 10% glycerol, 0.1 mM DTT and pH 8.0,) containing 8 M urea and incubated in 4°C with continuous stirring for 24 h to solubilize the IBs. Any insoluble material was removed by centrifugation at 4,000 rpm at 4°C for 1 h. The solublized IBs were refolded by 10-fold dilution into the buffer containing 50 mM Tris–HCl, 100 mM NaCl, 10 mM EDTA, 0.1 mM DTT and pH 8.0. The refolded enzyme was applied in purification experiments. 14 Chromatography methods were performed on a fast performance liquid chromatography (FPLC) system (Sykam, Germany). Firstly, the sample underwent anion exchange chromatography in a DEAE–Toyoperal 650 M column (3.0 cm × 15.0 cm) utilizing 0.1 M sodium phosphate buffer (pH 7.4) with the addition of 0.1 M NaCl, and a flow rate of 0.5 mL/min. The enzyme fractions were combined, then concentrated by ultrafiltration using Amicon Ultra centrifugal filter units (10,000 MWCO, 50 mL; Millipore, Billerica, MA, USA), and applied on a Sephadex G-100 (2.5 cm × 120 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.4). 33 At last, purified recombinant SK was sterilized by filtering through a 0.45 µm PVDF syringe filter, and the level of endotoxin was measured with LAL QCL 1000-TM Kit (LONZA, USA). 2.3. Activation of PSA PSAs with average MWs of 10.0 and 39.0 kDa were activated by oxidation of carbon 7 at the non-reducing end of the saccharide using periodate. To activate PSAs, a solution of 0.1 M sodium metaperiodate (NaIO 4 ) was combined with PSAs (10 mg PSA/mL NaIO 4 ) at 20°C, and the mixture was stirred with a magnetic stirrer for 15 minutes in the absence of light. Next, a two-fold volume of ethylene glycol was added to the reaction mixture, and was stirred for 30 minutes at 25°C. The oxidized PSAs were then precipitated using 70% cold ethanol, centrifuged at 15,000 rpm for 25 minutes at 20°C, and the resulting pellets were dissolved in a small amount of water before being stored at -20°C for future use. 27 , 28 Fig. 1 depicts the schematic illustration of PSA oxidation, and conjugation reaction of recombinant SK with oxidized PSA. 2.4. Measurement of PSA oxidation Estimation of the PSAs oxidation was performed using 2,4 dinitrophenylhydrazine (2,4-DNPH), which forms soluble 2,4 dinitrophenyl-hydrazones when combined with carbonyl substances. For a qualitative analysis, both unoxidized and oxidized CAs were mixed with the 2,4-DNPH reagent, the mixtures were stirred, and then left to precipitate at 37°C until the formation of a crystalline solid was noted. A quantitative approach for measuring PSAs oxidation using 2,4 DNPH was employed using propionaldehyde as a reference. In this procedure, propionaldehyde was combined with of 2,4-DNPH, and allowed to incubate for 10 minutes at 37°C, after which 0.5 M NaOH was added, followed by an additional incubation at 37°C for 5 minutes. The resulting color intensity was assessed at 490 nm. 27 2.5. Preparation of polysialylated recombinant SK Recombinant SK was chemically bonded to oxidized PSAs (10.0 and 39.0 kDa) through reductive amination with the addition of NaCNBH 3 at a concentration of 0.8 mg/mL (Fig. 1 ). To determine the best molar ratio of PSA to SK for conjugation, different ratios were mixed in 0.75 M K 2 HPO 4 (pH 9.0) with NaCNBH 3 (0.8 mg/mL) at 25°C with magnetic stirring. Samples were taken at 0, 12, 24, and 48 h, and subjected to (NH 4 ) 2 SO 4 precipitation to reach 70% w/v saturation. The samples were centrifuged at 8,000 rpm at 4°C for 20 minutes, and the pellets containing polysialylated SK were reconstituted in 1 mL 0.15 M phosphate buffered saline (PBS; pH 7.4) buffer, and dialysed overnight at 4°C against 0.5 L of 0.15 M PBS buffer. The dialysates were filtered through a 0.45 mm filter to remove insoluble material, and then concentration and activity of SK were determined. Controls involved exposing the native SK to the conjugation process without PSA, according to the specified conditions. Conjugation yield was calculated according to following formula: Conjugation yield (%) = (activity of remaining enzyme after conjugation/activity of enzyme before conjugation) ×100 (Eq. 1) Polysialylated SK was further examined, and identified using various methods as listed below. 29 , 30 2.6. Enzyme assay and protein determination The amidolytic activity of both native SK and polysialylated SK was assessed with the synthetic peptide substrate S-2251. 25 µL of plasminogen (0.2 mg/mL) and 50 µL of SK sample were mixed, and incubated at 37°C for 10 minutes. Then, 50 µL of S-2251 chromogenic substrate (0.75 mg/mL) was introduced, and incubated for additional 20 minutes at 37°C. 12,34 To halt the reaction, 50 µL of 10% acetic acid was introduced, and the sample was examined at 405 nm using a microplate reader. One unit of SK was defined as the quantity of enzyme, which liquefies a standard clot of plasminogen at 37°C, and pH 7.5 for 10 minutes. Protein quantification was conducted with the Bradford technique employing Coomassie Brilliant Blue G-250. 2.7. Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to detect changes in the molecular size of SK upon polysialylation. Electrophoresis was run using a 10.0% SDS gel at a current of 15 mA for 5 h. The SK and its conjugates were electrophoresed at 15 mA for 5 h. The protein bands were colored with Coomassie Brilliant Blue R-250 dye, and destained by diffusing in a solution with 40% (v/v) methanol and 10% (v/v) acetic acid. 2.8. Size exclusion chromatography Native and polysialylated SKs were subjected to size exclusion chromatography in a Sephadex G-100 column (0.9 cm × 30.0 cm) using 0.1 M sodium phosphate buffer (pH 7.4) with a flow rate of 0.4 mL/min on a HPLC system (Sykam, Germany). 35 Eluent fractions were assayed for SK activity. In the column, RNA polymerase (160 kDa), lactate dehydrogenase (142 kDa), glutamate dehydrogenase (90 kDa), bovin serum albumin (66 kDa), and cytochrome c (12.4 kDa) were applied as the MW standards. The MWs for native and polysialylated variants were calculated according to the calibration curve. 2.9. Mass spectrometry analysis The accurate determination of MW was determined by Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. For MALDI-TOF analysis, 1.0 mg/mL protein was desalted by passing through a G-25 Sephadex column according to the manufacturer’s instructions. The desalted protein samples were then spotted on MALDI plate, mixed with an equal volume of matrix solution of sinapinic acid in 50.0% v/v acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), air dried, and analyzed with a MALDI-TOF/TOF mass spectrometer instrument (Applied Biosystems 4800 MALDI TOF/TOF), operated in the positive ion mode. At last, the data were interpreted and processed using the instrument software (Applied Biosystems, USA). 14 2.10. Circular dichroism (CD) The Aviv-215 CD spectropolarimeter was used to record the spectra in 75.0 mM K 2 HPO 4 (pH 9.0) with protein solutions at a concentration of 0.3 mg/mL. A 1 cm cell path length was used for measuring from 190 to 260 nm (measurement parameters: bandwidth 1 mm, scanning speed 100 nm/min, response time 1.0 second). Following the correction stage which included a no-enzyme blank with 75.0 mM K 2 HPO 4 (pH 9.0) buffer, the spectra were smoothed using the instrument software. The calculation of molar ellipticity [θM] is determined using the following formula: θM = θobs/(c·d) (Eq. 2) Where θobs represents the observed ellipticity, c is the concentration of the enzyme [mol/L], and d is the path length of the quartz cuvette [cm]. At last, the wavelength was used to create a plot of θM, and the software of device was used to analyze the secondary structure content. 2.11. Intrinsic fluorescence spectroscopy A Cary Eclipse spectrometer (Varian, Australia) was used to measure intrinsic Trp fluorescence emission spectra. The excitation wavelength was set at 280 nm to selectively stimulate Trp, and the emission spectra were measured from 290 to 400 nm. Samples were at a concentration of approximately 100 µM. The protein samples were diluted to 100 µM in 75.0 mM K 2 HPO 4 buffer (pH 9.0). For the reagent blank, the same phosphate buffer was scanned under the same conditions. 2.12. Effect of polysialylation on kinetics parameters and thermal stability K m and V max of native and modified SK were calculated by the Hanes-Woolf plot using GraphPad Prism 7 (GraphPad Software, La Jolla California USA) with different concentrations of plasminogen (0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 and 3.0 mg/mL). The substrate concentration divided by the reaction rate ([S]/V) was graphed against substrate concentration ([S]) with the abscissa intercept, ordinate intercept and slope being K m , K m / V max and 1/ V max , respectively. For thermal stability test, the effect of temperature on the enzymatic reaction was analyzed by performing the activity test at various temperatures ranging from 20 to 50°C. The reaction mixture was pre-incubated at the specified temperatures for a total of 60 minutes before performing the activity assay. 2.13. Immunization evaluation of polysialylated SK Approval for the animal experiment was granted by the Animal Ethics Committee at Qazvin University of Medical Science under the code IR.QUMS.AEC.1402.009. Animals were executed by administrating carbon dioxide (CO 2 ) at the end of experiments. Male Wistar rats weighing 250–300 g were acquired from the Pasteur Institute of Iran in Karaj. The rats were housed in a setting at 22°C with a relative humidity of 55%, and light/dark cycles (12 h/12 h) with food and water available throughout the study. The rats were divided into three groups (n = 5) as follows: 1- Negative control (NC) group received 1 mL of normal saline. 2- Native group received native SK. 3-Polysialylated group received polysialylated SK. Each rat of native or polysialylated groups was given a dose of 5000.0 IU (0.2 mg/mL) of sterile solution of either native or polysialylated SK through the tail vein. Rats were immunized, and blood samples were taken on days 7, 14, and 21. The blood was then centrifuged at 4,000 rpm for 20 minutes at 25°C to obtain antiserum, which was stored at -30°C. Antibody titers in the rats were analyzed using an indirect enzyme-linked immunosorbent assay (ELISA). Polystyrene plates were covered with 100 µL of coating solution (50 mM sodium carbonate, pH 9.6) with 5 µg/mL of native or polysialylated SK in each well, then left to incubate overnight at 4°C. The wells were rinsed three times with 300 µL of PBS containing 0.1% Tween 20 (PBS-T). The unoccupied sites were blocked by adding 200 µL of PBS-T containing 3.0% BSA, and incubated for 2 h at 20°C. The wells were washed with PBS-T, and incubated with 300 µL of serum serially diluted in PBS-T containing 1% BSA at 20°C for 2 h. The wells were again washed and tap dried. Next, 100 µL of sheep anti-rat IgG-peroxidase conjugate (diluted to 1:750 in PBS-T with 1% BSA) was added to each well, followed by a 2 h incubation at 20°C. The unbound antibodies were removed by washing the wells with PBS-T, and 100 µL of tetramethylbenzidine (TMB) substrate solution was added. Following 30 min incubation at 25°C, the reaction was halted by introducing 50 µL of 1.0 N H 2 SO 4 soultion, and the absorbance was measured at 450 nm. Negative control wells without SK enzyme or diluted rat serum were included in each run. The half maximal effective concentration (EC 50 ) was calculated according to a nonlinear regression curve fit with increasing concentrations of native or polysialylated SK. 2.14. Plasma half-life of polysialylated SK Male Wistar rats weighing between 250 and 300 g were housed in a controlled environment set at 22°C with 55% relative humidity, under a 12 h light/dark cycle, and provided with food and water throughout the study. Each group of rats (N = 5) was anesthetized using ketamine and xylazine, and a warm water bag was used to induce mild vasodilation in the tail. The rats were divided into three groups (n = 5) as follows: 1- Negative control (NC) group received 1 mL of normal saline. 2- Native group received native SK. 3-Polysialylated group received polysialylated SK. Approximately 7500.0 IU (0.34 mg/mL) of either native SK or polysialylated variant in sterile solution was administered to rats through the tail vein. Around 50 µl of complete blood samples were taken from rats through tail transection at 10, 20, 30, 60, 120, 180, and 360 minutes following drug administration. The blood samples were put into heparinized Eppendorf tubes and centrifuged at 3,000 rpm for 10 minutes at 25°C to separate plasma, then stored at -20°C. SK activity in plasma was measured using a colorimetric method as mentioned before. 2.15. Statistical analysis All tests were carried out at least three times, and each value represented the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software, La Jolla California USA) to compare the statistical significance between the native and polysialylated variants. Differences with p -values below 0.05, and confidence levels of 95.0% were considered significant. 3. Results 3.1. Production of recombinant SK enzyme For bacterial production of SK enzyme, a recombinant plasmid based on the expression vector pEKG-3 was constructed. The synthetic gene of SK was assembled based on the E. coli W3110 codon usage. PCR amplification of codon-optimized SK generated a 1248 bp fragment. The obtained PCR product was cut with Eco RI and Bam HI restriction enzymes, purified from the gel, and ligated into pEKG-3 vector. The recombinant plasmid was confirmed by restriction analysis, and DNA sequencing, and then transferred into E. coli W3110 for expression. The purified refolded recombinant SK enzyme was duly detected by SDS-PAGE. The SK migrated at approximately 47.0 kD by SDS-PAGE (Fig. 2 ). The yield of enzyme production and biomass concentration under the optimized conditions were estimated to be 19.0% of total protein, and 17.5 g per 1 L culture medium, respectively. 3.2. Synthesis and characterization of recombinant polysialylated SK To obtain the optimal condition for enzyme polysialylation, CAs (10.0 and 39.0 kDa) were coupled to SK at 50:1, 100:1, 150:1, and 200:1 PSA: SK molar ratios. The reactions were followed by ammonium sulfate precipitation, and conjugation yields were calculated based on the PSAs found in the precipitate (Fig. 3A). As seen, the highest conjugation yield (64.52%) was achieved by 10.0 kDa PSA at molar ratio of 200:1. Thereafter, the influence of incubation time (Fig. 3B) and temperature (Fig. 3C) on the conjugation yield was also investigated. The results showed that 24 h and 25°C were the best time, and temperature for conjugation reaction, respectively. The purified native SK migrated at approximately 47.0 kD by SDS-PAGE (Fig. 4 ). When SDS-PAGE was applied to detect changes in the MW of SK, polysialylated SKs exhibited different electrophoresis migration at around 55.0 kDa and 80.0 kDa, which confirmed the conjugation of SK to 10.0 and 30.0 kDa PSAs, respectively (Fig. 4 ). However, according to the low conjugation yield (21.23%), faint band of SK-39.0 kDa PSA variant compared to the SK-10.0 kDa PSA at equivalent protein concentrations in SDS-PAGE (Fig. 4 ) as well as significant loss of activity of SK-39.0 kDa PSA (results not shown), we decided to do the next experiments only with SK-10.0 kDa PSA variant. The samples were further analyzed using size exclusion chromatography in a Sephadex G-100 column (Fig. 5 ). As observed, a peak of native SK was detected at approximately 12 minutes (Fig. 5 A), whereas polysialylated SK eluted at approximately after approximately 22 minutes of injection (Fig. 5 B) indicating the higher MW of the polysialylated variant. The exact MWs of native SK and SK-10.0 kDa PSA were determined by MALDI-TOF mass spectrometry (Fig. 6 ). The data showed an exact MW of 56.5 kDa for SK-10.0 kDa PSA (Fig. 6 B) which nearly matches the calculated value by SDS-PAGE. The monodisperse composition was confirmed for both samples (just one peak) with no detectable shoulders or minor peaks related to impurities or truncated forms. Conjugation reaction was also analyzed by CD and fluorescence spectroscopy methods. CD spectra showed that the polysialylation induces a decrease in the far UV CD signal, suggesting an increase in the protein alpha helix content (Fig. 7 A). The intrinsic Trp fluorescence intensity of polysialylated SK increased compared to the native version (Fig. 7 B), indicating changes in the microenvironments of Trp residues. 3.3. Kinetic parameters and thermal stability To evaluate the effect of polysialylation on the enzyme’s kinetics, kinetics data were determined based on the initial velocity measurements of the S-2251 degradation (Fig. 8 A). In the present study, K m values estimated by the Hanes Woolf plot for native and polysialylated SKs were 31.1 and, 32.5 mg/mL, respectively. From the same plot, calculated V max values for native and polysialylated enzymes were 283.5, and 294.2 µmol min − 1 , respectively. The thermal stability of polysialylated variant was also studied. As observed (Fig. 8 B), polysialylated SK retained its activity up to 91.0% at 55.0°C while native enzyme lost nearly 50.0% of original activity at this temperature. 3.4. Evaluation of immunization and pharmacokinetic of polysialylated SK Results (total IgG) in Table 1 indicate that, at equivalent protein concentrations, polysialylated SK elicited nearly 62.0% lower antibody production compared to the native variant. Moreover, the lower value (16000.0 IU ± 65.0) of SK-10.0 kDa PSA concentration (Table 1 ) that achieved EC 50 exhibited that polysialylated SK was more potent compared to the native enzyme. The in vivo pharmacokinetics of native SK and polysialylated variant were also assessed. The enzyme activity was significantly ( p < 0.03) higher in the rats injected with polysialylated SK than in those injected with native enzyme (Fig. 9 ). The comparison of pharmacokinetics parameters estimated by statistical analysis is also depicted in Table 2 . Polysialylation of SK yielded preparation with a longer plasma half-life (2.21 h) compared to the native variant (0.5 h). Table 1 Effects of polysialylation on IgG titer, and half-maximal effective concentration (EC 50 ). Antisera raised against native and polysialylated streptokinase (SK). The significant difference between each of the coatings with polysialylated SK to the coating with native enzyme was p < 0.01. Variant Total IgG titer (OD) (Mean ± SD) 7 Days 14 Days 21 Days EC 50 (IU) Native 320 ± 12.0 1120 ± 45.0 1750 ± 55.0 26000.0 IU ± 50.0 Polysialylated 120 ± 8.5 420 ± 35.0 690 ± 42.0 16000.0 IU ± 65.0 All values are reported as the mean ± standard deviation (SD) of three measurements. Table 2 Comparison of pharmacokinetics parameters of native streptokinase (SK) and polysialylated variant. SK variant Half-Life (h) Volume of distribution (L/kg) Clearance (L/hr/kg) Native 0.5 ± 0.21 23.33 ± 2.1 38.22 ± 3.2 Polysialylated 2.21 ± 0.32 13.55 ± 4.5 6.46 ± 5.3 The values represent mean ± standard deviation of two independent experiments. P < 0.02: significant difference vs. native variant. 4. Discussion Polymer-based drug delivery systems have been utilized in biomedical applications in attempts to improve the therapeutic activity of drug materials. Potential advantages of these delivery mechanisms include an increased or prolonged duration of pharmacologic activity, a decrease in adverse effects, increased patient compliance and quality of life. 36 , 37 Among the available techniques, polymer–drug conjugates have exhibited clinical and commercial success in the field of drug delivery. Polymer-drug conjugates are a part of therapeutics, in which the therapeutic agent is chemically linked to a polymer instead of being encapsulated into it. They offer distinct features, such as reduced drug toxicity, increased drug solubility, enhanced drug bioavailability, biocompatibility, reduced immunogenicity, and improved pharmacokinetic parameters. Such strategies have the potential to develop as the next generation protein therapeutics. Over the past decade, numerous polymer–drug conjugates-based formulations entered the market, and several others are in clinical trials. 38 – 40 PEG is among the most well-understood and utilized synthetic polymers, and has been FDA approved as a material for polymer–drug conjugates. Grafting of hydrophilic macromolecules such as PEG onto the surface of drugs has proved successful in prolonging presence in blood circulation, and reducing immunogenicity with consequent increase of therapeutic efficacy. 14,18 However, conjugation of PEG to enzymes often leads to substantial reduction of their activity. Moreover, the non-biodegradable PEG is expected to accumulate in the lysosomes following endocytosis of the conjugates, possibly leading to toxicity on chronic use. Despite various successes of PEGylation, some products were withdrawn from the market because of polymer toxicity or limited drug loading. To overcome the major issues of biodegradability and toxicity, natural polymers, such as PSA is being explored as a safe and alternative solution. 41 Polysialylation with the use of the highly hydrophilic and biodegradable polymer of PSA as an alternative to PEG in prolonging the circulatory half-lives of proteins has been suggested. PSA can be used to protect therapeutic molecules from the host immune system, and improve their pharmacokinetics. 20 , 23 , 42 SK is a highly effective thrombolytic agent, and often used in less affluent societies for the myocardial infarction. 43 , 44 However, if some of its major drawbacks can be rectified, it has the potential to occupy centre-stage as an effective thrombolytic, especially for ischemic strokes. 2–4 Hence, several studies have tried to increase half-life, minimize immunogenicity and improve the biological efficacy with different mechanisms, such as protein engineering and developing delivery systems for SK. 8,16,17 It has been shown that PEGylation of SK considerably enhances its therapeutic attributes. 14 The production of antibodies against PEGylated drugs has also been reported in some cases, and this proves to be a great disadvantage for PEG applications. 21 So, there is a need to find a biocompatible polymer for conjugation to SK that can reduce the immunogenicity without compromising its enzymatic activity. In the present investigation, polysialylation of SK was attempted through in vitro chemical conjugation. For bacterial production of SK enzyme, a recombinant plasmid based on the generic expression vector pEKG-3 was constructed. The recombinant expressed SK migrated at approximately 47.0 kD by SDS-PAGE. In polysialylation of enzyme, the highest conjugation yield (64.52%) was achieved by 10.0 kDa PSA and 200:1 molar ratio. The results showed that 24 h and 25°C were the best time and temperature for conjugation reaction, respectively. Initial evidence that SK was covalently linked to PSA to form a sialyated enzyme was obtained by SDS-PAGE. The polysialyated SKs exhibited different electrophoresis migrations at around 55.0 kDa and 80.0 kDa which confirmed the conjugation of SK to PSA (Fig. 4 ). However, according to the low conjugation yield (21.23%), faint band of SK-39.0 kDa PSA variant compared to the SK-10.0 kDa PSA at equivalent protein concentrations in SDS-PAGE (Fig. 4 ) as well as significant loss of activity of SK-39.0 kDa PSA (results not shown), we decided to do the next experiments only with SK-10.0 kDa PSA variant. It seems that PSA with higher MW such as 30.0 kDa is more suitable for the small protein or peptides. This is supported by work on insulin polysialylation in which 39.0 kDa PSA was optimized for conjugation. 26 In contrast, for the several other proteins, e.g., asparaginase, 30 catalase, 28 and uricase, 32 10.0 kDa PSA has been utilized. The exact MWs of native SK and SK-10.0 kDa PSA were determined by MALDI-TOF mass spectrometry (Fig. 6 ). Conjugation reaction was also analyzed by CD and fluorescence spectroscopy. CD spectra showed that polysialyation induces a decrease in the far UV CD signal which suggests an increase in the protein alpha helix content (Fig. 7 A). This finding implies that polysialyated SK has greater protein stability than the native version. Structural stability and conformational state of polysialylated enzyme was further analyzed by measuring the native intrinsic Trp fluorescence emission at 290 nm, while being excited from 290 to 400 nm. Intrinsic protein fluorescence deriving from the naturally fluorescent Trp can provide information on the conformational changes of proteins, and their interactions with other molecules. 45 , 46 The intrinsic Trp fluorescence intensity of polysialylated SK increased compared to the native version (Fig. 7 B), indicating changes in the microenvironments of Trp residues. As a general rule, in the folded state, Trp residues are mainly located in the hydrophobic core of proteins. When they become exposed to solvents (a hydrophilic environment) in an unfolded state, they will have reduced fluorescence intensity. 47 Thus, it can be said that the increment of fluorescence intensity in polysialylated enzyme means that stability of SK was increased by immobilizing on PSA polymer, consistent with the CD results. On the other hand, there was no notable difference in the maximum fluorescence emission wavelength (λmax) between native and polysialylated SK, i.e., no unfavorable conformational changes in SK polysialylation are occurred. The interpretation of the fluorescence spectroscopy leads to the conclusion that polysialylation of SK not only changes the conformational state, but also stabilizes the enzyme structure. Chemical modification of enzymes with polymers is known to often adversely affect their kinetic parameters, and thus limit their efficacy. 23 Evidently, a prime consideration in employing polysialylation for enzymes is the maintenance of their function. In the present study, K m values estimated by the Hanes Woolf plot for native and polysialylated SKs were 31.1 and, 32.5 mg/mL, respectively. From the same plot, calculated V max values for native and polysialylated enzymes were 283.5, and 294.2 µmol min − 1 , respectively. As can be found, K m of polysialylated SK is slightly higher than that of native SK, showing that the attached PSA molecule does not significantly affect the enzyme affinity toward its substrate. It is likely that this phenomenon was due to the little conformational change of SK on PSA attachment, rather than to the diffusional barrier of PSA. In other words, it means that, PSA polymer had not significant structural influence on the active site of SK. This result might also be attributed to the limited number of PSAs bound per molecule of enzyme. Taken together, the kinetic results indicate that the covalent coupling of PSA to SK does not affect its substrate specificity, and the polysialylated enzyme will be able to effectively act in vitro. Similar results have been obtained for the polysialylation of other enzymes, e.g., catalase, 28 uricase 32 and asparginase. 30 The thermal stability of polysialylated variant was also studied. As observed (Fig. 8 B), polysialylated SK retained its activity up to 91.0% at 55.0°C while native enzyme lost nearly 50.0% of original activity at this temperature. The higher activity of polysialylated SK at temperature of 55°C after 1 h of incubation suggests that polysialylation has improved the overall stability of the enzyme. Indeed, this is an interesting advantageous for any pharmaceutical application. Unwanted immunogenicity of conjugated proteins to polymers (e.g., PEG or PSA) can cause undesirable side effects, and render them ineffective. The immune-reactivity of polysialylated SK and native variant was measured by indirect ELISA using antisera raised against each of the antigen preparations. Results (total IgG) indicate that, at equivalent protein concentrations, polysialylated SK elicited nearly 62.0% lower antibody production compared to the native variant. Our interpretation of these findings is that the conjugated PSA chains sterically hinder the binding of IgG molecules to the relevant antigenic sites on SK, leading in turn to reduce antigenicity. In other words, PSA chains may help to mask some antigenic determinants (epitopes), and prevent complexing of SK with its antibodies. This was in line with the finding that immune complexes are formed by spatial complementarity, and that the forces that bind antigen and antibody together are weakened by an increased distance between the two molecules. 48 Another reason that may have contributed to the reduction of antigen–antibody binding would be the generation of repulsive forces due to the negative charge of the enzyme-bound PSAs. Moreover, it is conceivable that loss of some of the free-amino groups of SK on polysialylation has rendered the modified enzyme intrinsically more negatively charged. In accordance with our results, similar reductions in immunogenicity with polysialylation have been reported for asparginase, 29 insulin, 27 erythropoietin, 42 uricase, 32 and catalase. 28 The in vivo pharmacokinetics of native SK and polysialylated variant were also assessed. The enzyme activity was significantly ( p < 0.03) higher in the rats injected with polysialylated SK than in those injected with native enzyme (Fig. 9 ). The comparison of pharmacokinetics parameters estimated by statistical analysis is also depicted in Table 2 . Polysialylation of SK yielded preparation with a longer plasma half-life (2.21 h) compared to the native variant (0.5 h). In comparison, half-life of polysialylated SK is longer than the reported values for encapsulated SK in mPEG-PLGA (2.0 h), 13 PEG-grafted chitosan/SK nanoparticles (2.0 h), 12 and truncated PEGylated SK (2.0 h). 14 Indeed, the increased period of pharmacological activity of polysialylated SK suggests that polysialylation contributes to longer retention of enzyme activity. The similar results with catalase, 28 insulin, 27 asparaginase, 29 interferon α-2b 23 and several other proteins strongly show that the presence of PSA chains on the molecules allows retention of much the activity in polysialylated therapeutics. 5. Conclusions In summarize, the data presented in this paper reveals that the enhanced properties obtained by SK-10.0 kDa PSA, particularly structural stability, reduced immunogenicity, and extended half-life can contribute to ameliorate the therapeutic value of this thrombolytic drug. It is noteworthy that polysialylation technology offers a promising strategy for the synthesis of a novel version of SK with improved pharmacological properties. In the meanwhile, since this research is our preliminary data, further studies is required to confirm the efficacy of this formulation on human. In light of this, we plan to continue our work with further optimization of conjugation reaction, additional preclinical studies, and human clinical trials of polysialylated SK. Declarations AUTHOR INFORMATION Corresponding Author Hamid Shahbazmohammadi − Cellular and Molecular Research Center, Research Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences , Qazvin, Iran ; https://orcid.org/0000-0002-2099-1812; Email: [email protected] Authors Eskandar Omidinia − Enzyme Technology Laboratory, Department of Biochemistry, Genetic and Metabolism, Research Group, Pasteur Institute of Iran, Tehran, Iran ; https://orcid.org/0000-0002-3002-5714 Author Contributions Hamid Shahbazmohammadi: Writing–review & editing, Conceptualization, Methodology, Formal analysis. Eskandar Omidinia : Supervision, Project administration, Funding acquisition. Notes The authors declare no competing financial interest. Acknowledgements This study was supported by the Qazvin University of Medical Sciences (project number: 402000043). References Kiran GR, Chandrasekhar P, Mohammad Ali S. Clinical outcomes of patients with mitral prosthetic valve obstructive thrombosis treated with streptokinase or tenecteplase. Indian Heart J. 2021;73:365–8. Babbal A, Mohanty S, Khasa YP. Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris : a case study with proteolysis prone streptokinase. Int J Biol Macromol. 2021;180:760–70. Locke M, Rigsby P, Longstaff C. An international collaborative study to establish the WHO 4th international standard for streptokinase: communication from the SSC of the ISTH. J Thromb Haemost. 2020;181:501–1505. Ayinuola YA, Brito-Robinson T, Ayinuola O, Beck JE, Cruz-Topete D, Lee SW, Ploplis VA, Castellino FJ. Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence. J Biol Chem. 2021;296:100099. Mahmoud AAA, Mahmoud HE, Mahran MA, Khaled M. Streptokinase versus unfractionated heparin nebulization in patients with severe acute respiratory distress syndrome (ARDS): a randomized controlled trial with observational controls. J Cardiothorac Vasc Anesth. 2020;34:436–43. El-Dabaa E, Okasha H, Samir S, Nasr SM, El-Kalamawy HA, Saber MA. Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity. Arab J Chem. 2022;15:103799. Dey S, Guchhait KC, Manna T, Panda AK, Patra A, Mondal SK, Ghosh C. Evolutionary and compositional analysis of streptokinase including its interaction with plasminogen: an in silico approach. Gene Rep. 2022;29:101689. Taheri MN, Behzad-Behbahani A, Rafiei Dehbidi G, Salehi S, Sharifzadeh S. Engineering, expression and purification of a chimeric fibrin-specific streptokinase. Protein Expression Purif. 2016;128:14–21. Mehrad H, Foletti A. Effect of optison microbubbles-mediated B-mode ultrasound- guided focused low-level confocal dual pulse electrohydraulic shock wave-assisted streptokinase thrombolysis on embolic carotid artery. Atherosclerosis. 2021;331:e263. Mulpuri VB, Vaswani P, Gupta R, Rana SS, Kang M, Gorsi U, Gupta R. Fr298 streptokinase has a role in salvaging patients undergoing step up treatment who fail large volume saline lavage. Gastroenterol. 2021;160:S–289. Bhargava V, Gupta R, Vaswani P, Jha B, Rana SS, Gorsi U, Kang M, Gupta R. Streptokinase irrigation through a percutaneous catheter helps decrease the need for necrosectomy and reduces mortality in necrotizing pancreatitis as part of a step-up approach. Surgery. 2021;170:1532–7. Baharifar H, Khoobi M, Arbabi Bidgoli S, Amani A. Preparation of PEG-grafted chitosan/streptokinase nanoparticles to improve biological half-life and reduce immunogenicity of the enzyme. Int J Biol Macromol. 2020;143:181–9. Hasanpour A, Esmaeili F, Hosseini H, Amani A. Use of mPEG-PLGA nanoparticles to improve bioactivity and hemocompatibility of streptokinase: in-vitro and in-vivo studies. Mater Sci Eng C. 2021;118:111427. Sawhney P, Katare K, Sahni G. PEGylation of truncated streptokinase leads to formulation of a useful drug with ameliorated attributes. PLoS ONE. 2016;11:e0155831. Mehrad H, Shokouhi MF. Sonothrombolysis of embolic carotid artery, using pesda microbubbles- mediated B-mode ultrasound- guided extracorporeal pulsed low- level focused confocal dual frequency ultrasound accompanied by streptokinase administration. Atherosclerosis. 2021;331:e264. Adivitiya, Khasa YP. The evolution of recombinant thrombolytics: current status and future directions. Bioengineered. 2017;8:331–58. Sevostyanov MA, Baikin AS, Sergienko KV, Shatova LA, Kirsankin AA, Baymler IV, Shkirin AV, Gudkov SV. Biodegradable stent coatings on the basis of PLGA polymers of different molecular mass, sustaining a steady release of the thrombolityc enzyme streptokinase. React Funct Polym. 2020;150:104550. Sawhney P, Kumar S, Maheshwari N, Guleria SS, Dhar N, Kashyap R, Sahni G. Site-specific thiol-mediated PEGylation of streptokinase leads to improved properties with clinical potential. Curr Pharm Des. 2016;22:5868–78. Torréns I, Ojalvo AG, Seralena A, Pupo E, Lugo V, Páez R. A Mutant streptokinase lacking the C-terminal 42 amino acids is less reactive with preexisting antibodies in patient sera. Biochem Biophys Res Commun. 1999;266:230–6. Pelingon R, Pegg CL, Zacchi LF, Phung TK, Howard CB, Xu P, Hardy MP, Owczarek CM, Schulz BL. Glycoproteomic measurement of site-specific polysialylation. Anal Biochem. 2020;596:113625. Najjari A, Shahbazmohammadi H, Nojoumi SA, Omidinia E. PASylated urate oxidase enzyme: enhancing biocatalytic activity, physicochemical properties, and plasma half-life. ACS Omega. 2022;7:46118–30. Constantinou A, Chen C, Deonarain MP. Polysialic acid and polysialylation to modulate antibody pharmacokinetics, in therapeutic proteins. Wiley Online Libaray; 2012. pp. 95–115. Gregoriadis G, Jain S, Papaioannou I, Laing P. Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Int J Pharm. 2005;300:125–30. Lindhout T, Iqbal U, Willis LM, Reid AN, Li J, Liu X, Moreno M, Wakarchuk WW. Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proc. Natl. Acad. Sci. 2011, 108, 7397–7402. Zhang T, Zhou S, Hu L, Peng B, Liu Y, Luo X, Liu X, Song Y, Deng Y. Polysialic acid-polyethylene glycol conjugate-modified liposomes as a targeted drug delivery system for epirubicin to enhance anticancer efficiency. Drug Deliv Transl Res. 2018;8:602–16. Smirnov IV, Vorobiev II, Belogurov AA, Genkin DD, Deyev SM, Gabibov AG. Chemical polysialylation of recombinant human proteins, in glyco-engineering: methods and protocols, Castilho A. Editor. 2015, Springer, New York, 2015, pp. 389–404. Jain S, Hreczuk-Hirst DH, McCormack B, Mital M, Epenetos A, Laing P, Gregoriadis G. Polysialylated insulin: synthesis, characterization and biological activity in vivo. Biochim Biophys Acta-Gen Subj. 2003;1622:42–9. Fernandes AI, Gregoriadis G. Synthesis, characterization and properties of sialylated catalase, Biochim. Biophys. Acta. Protein Struct Mol Enzymol. 1996;1293:90–6. Fernandes AI, Gregoriadis G. The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics. Int J Pharm. 2001;217:215–24. Fernandes AI, Gregoriadis G. Polysialylated asparaginase: preparation, activity and pharmacokinetics. Biochim Biophys Acta Protein Struct Mol Enzymol. 1997;1341:26–34. Zhang T, Zhou S, Hu L, Peng B, Liu Y, Luo X, Song Y, Liu X, Deng Y. Polysialic acid-modifying liposomes for efficient delivery of epirubicin, in-vitro characterization and in-vivo evaluation. Int J Pharm. 2016;515:449–59. Punnappuzha A, PonnanEttiyappan JB, Sekhar Nishith R, Hadigal S, Ganapathi Pai P. Synthesis and characterization of polysialic ccid–uricase conjugates for the treatment of hyperuricemia. Int J Pept Res Ther. 2014;20:465–72. Shahbazmohammadi H, Omidinia E. Development and application of aqueous two-phase partition for the recovery and separation of recombinant phenylalanine dehydrogenase. Iran J Chem Chem Eng. 2008;27:119–27. Shahbazmohammadi H, Omidinia E, Sahebghadam Lotfi A, Saghiri R. Preliminary report of NAD + -dependent amino acid dehydrogenase producing bacteria isolated from soil. Iranianian Biomed J. 2007;11:131–5. Shahbazmohammadi H, Omidinia E, Dinarvand R, Taherkhani HA. Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase. Bioprocess Biosyst Eng. 2010;33:317–29. Alven S, Nqoro X, Buyana B, Aderibigbe BA. Polymer-drug conjugate, a potential therapeutic to combat breast and lung cancer. Pharmaceutics. 2020;12:406. Thakor P, Bhavana V, Sharma R, Srivastava S, Singh SB, Mehra NK. Polymer–drug conjugates: recent advances and future perspectives. Drug Discov Today. 2020;25:1718–26. Pisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J Pharm Sci. 2010;99:2557–75. Ekladious I, Colson YL, Grinstaff MW. Polymer–drug conjugate therapeutics: advances, insights and prospects. Nat Rev Drug Discovery. 2019;18:273–94. Larson N, Ghandehari H. Polymeric conjugates for drug delivery. Chem Mater. 2012;24:840–53. Wu J, Lu S, Zheng Z, Zhu L, Zhan X. Modification with polysialic acid–PEG copolymer as a new method for improving the therapeutic efficacy of proteins. Prep Biochem Biotechnol. 2016;46:788–97. Meng H, Jain S, Lockshin C, Shaligram U, Martinez J, Genkin D, Hill DB, Ehre C, Clark D, Hoppe. H.Clinical application of polysialylated deoxyribonuclease and erythropoietin. Recent Pat Drug Deliv Formul. 2018;12:212–22. Alanazi AZ, Alhazzani K, Mostafa AM, Barker J, El-Wekil MM, Ali A. M.B.H. Highly selective fluorometric detection of streptokinase via fibrinolytic release of photoluminescent carbon dots integrated into fibrin clot network. Microchem J. 2024;197:109800. Turkia HB, Souissi S, Chabaane F, Chermiti I, Fadhel R, Keskes S, Bekir A, Ghazali H. 172 Fibrinolysis of ST elevation myocardial infarction in emergency department: prognosis of elderly patients treated with streptokinase. Resuscitation. 2024;203:S88. dos Rodrigues S, Delgado FH, Santana da Costa GG, Tasic T. Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts. BBA Adv. 2023;3:100091. Gooran N, Kopra K. Fluorescence-based protein stability monitoring-a review. Int J Mol Sci. 2024;25:1764. Cao Y, Zhao J. Innovative application of Coomassie Brilliant Blue: a simple, economical, and effective determination of water-insoluble protein surface hydrophobicity. Anal Methods. 2015;8:790–5. Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitts’s Essential Immunology. 8th ed. Oxford, UK: Blackwell Scientific; 1994. pp. 81–102. Additional Declarations No competing interests reported. Supplementary Files GA.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7604522","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":523712406,"identity":"59f2f994-cd23-45b3-a41c-0ac042d4e5ad","order_by":0,"name":"Hamid Shahbazmohammadi","email":"data:image/png;base64,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","orcid":"","institution":"Qazvin University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Shahbazmohammadi","suffix":""},{"id":523712407,"identity":"3d06b304-d86a-4820-b634-73120a186f0f","order_by":1,"name":"Eskandar Omidinia","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Eskandar","middleName":"","lastName":"Omidinia","suffix":""}],"badges":[],"createdAt":"2025-09-13 04:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7604522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7604522/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92728050,"identity":"bb9cdbc2-ea83-4836-bb52-49cdd3231c5c","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3267072,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptSK.doc","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/4626ae34b0a78aa0818745b8.doc"},{"id":92728028,"identity":"7358bf9b-370b-41de-82d0-54a2c12e5011","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5365,"visible":true,"origin":"","legend":"","description":"","filename":"6bbc11b636904d7a843b784d638fd95c.json","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/348b54ba1219432f14678025.json"},{"id":92728022,"identity":"b7c11288-dc67-4383-821a-f6c41872850c","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141817,"visible":true,"origin":"","legend":"","description":"","filename":"6bbc11b636904d7a843b784d638fd95c1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/bfa6359e2b079f2416b95b55.xml"},{"id":92728023,"identity":"617a2542-0473-43ab-8b6d-d6be891e8a92","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"eps","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":539,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage1.eps","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/51e48dcdda8054c5cbd2ee4d.eps"},{"id":92728015,"identity":"517a3edf-c297-48af-b986-4ce59d9e17a4","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"eps","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48596,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage2.eps","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/f6e6fab101c0a0bd58d1e1f6.eps"},{"id":92728038,"identity":"eb3e5878-7eee-4d2a-a8af-b988ff93d81b","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"eps","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48596,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage2.eps","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/0a68898b3684fd3b0a14fde3.eps"},{"id":92728013,"identity":"04e2c604-f520-4759-a802-3bf5afcf33de","added_by":"auto","created_at":"2025-10-03 15:01:58","extension":"eps","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48596,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage2.eps","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/1fa6fb3902c9be324e0bd448.eps"},{"id":92728042,"identity":"d69c2eaf-1fa1-4005-bac8-80be113fe944","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"eps","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48596,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage2.eps","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/ed467a24183b188f0f109685.eps"},{"id":92728001,"identity":"273027d6-e86a-4127-9894-e50979dfb520","added_by":"auto","created_at":"2025-10-03 15:01:56","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":854494,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/948fdefa91a6709c590851e5.jpeg"},{"id":92728018,"identity":"b77605f2-9a64-434a-8644-8355c3152f7f","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":519934,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/eb98e5b36aa6154ab8020f05.jpeg"},{"id":92728033,"identity":"ead8b6e6-0800-4b0f-b179-5c7468b01ff3","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176797,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/7e21c11728e1e8957d86caf8.jpeg"},{"id":92728014,"identity":"cd5747a8-6761-4826-b6a7-65cae012073f","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":286618,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/fe16decc9247c5f71d5c2702.jpeg"},{"id":92728659,"identity":"a8c9a37c-09df-4d9b-9f7d-cf21beab9465","added_by":"auto","created_at":"2025-10-03 15:10:01","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":356176,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/20e36f3ffae8e3956cc61b58.jpeg"},{"id":92728654,"identity":"d8439a5f-8b76-4397-8c93-c665be7f63ba","added_by":"auto","created_at":"2025-10-03 15:09:57","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":260240,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/30ef2a23c5b6e34175ee1337.jpeg"},{"id":92728046,"identity":"28c4e7ce-4b93-4257-b1d5-f50d92930adc","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":214160,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/1733831dd3eff950efb5c348.jpeg"},{"id":92728051,"identity":"a3d23de8-2253-41a9-97ea-3d9de57f8114","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":255661,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/408db7e4628daaf83837c151.jpeg"},{"id":92728016,"identity":"e499b7e9-2f30-4fcb-88fd-696f7e6cdf91","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":285674,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/6e6ddcfcf38a952d41a4cb5e.jpeg"},{"id":92728029,"identity":"21904e19-61f1-4043-b8b9-c7f1062310be","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23143,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/e5418238e7693417e95415a8.jpeg"},{"id":92728666,"identity":"cdba1e1d-2df5-4349-b8c8-02870e41ad65","added_by":"auto","created_at":"2025-10-03 15:10:02","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18392,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/bf1f8fb8391a57356f9d9408.jpeg"},{"id":92728034,"identity":"06b28d29-1052-4cde-b9f2-5ca9c30ea664","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16659,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/949ac96f77b9c61fbcd6d651.jpeg"},{"id":92728021,"identity":"e47da4bd-0670-4ec0-af5d-1a8b793f97f7","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52461,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/c2022a7f3debb00485d7c637.jpeg"},{"id":92728653,"identity":"ada49634-34e7-4fb1-bca5-4428286f14fd","added_by":"auto","created_at":"2025-10-03 15:09:57","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37577,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/7a3cdb4177001b18cb890cd4.jpeg"},{"id":92728048,"identity":"eb077a40-9715-479f-ae49-e97fc7179a40","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"jpeg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33270,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/71edd40d015929d3881044c6.jpeg"},{"id":92728012,"identity":"1c3bcf29-ac49-4b2b-b09f-ca52c32cf794","added_by":"auto","created_at":"2025-10-03 15:01:58","extension":"jpeg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29806,"visible":true,"origin":"","legend":"","description":"","filename":"groupimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/a2b534e4a858c80a516a594c.jpeg"},{"id":92728056,"identity":"48448f72-baa0-4229-b504-d7c68f91fff4","added_by":"auto","created_at":"2025-10-03 15:02:02","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159548,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/4a0f34db5e02f8233f64557a.png"},{"id":92728032,"identity":"dd331c94-4803-477f-aec9-8659792f663d","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91683,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/daa522a1448cb35f4390b217.png"},{"id":92728664,"identity":"7cd55b9f-de8c-4ce1-ab11-352aabaeffa8","added_by":"auto","created_at":"2025-10-03 15:10:01","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40044,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/ee70c7cb8cc7ca872e1c3719.png"},{"id":92728010,"identity":"d70c649b-bc43-4c48-ac8e-955c534348a8","added_by":"auto","created_at":"2025-10-03 15:01:58","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72614,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/508aeef9365c436b99a228a2.png"},{"id":92728025,"identity":"2e5c682d-eae8-4e3e-91a5-a386b31690c7","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68708,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/f8146ae6648313690682f8a8.png"},{"id":92728011,"identity":"924367f4-5a5c-4549-8d89-e697bc3b8636","added_by":"auto","created_at":"2025-10-03 15:01:58","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34744,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/fd8aabd3600e34eb90fe7176.png"},{"id":92728039,"identity":"230eeb53-63cd-4ff9-ad81-aa8b14696af0","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49086,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/070dff30596a2e4880bc88f6.png"},{"id":92728024,"identity":"c568cba2-d6ef-4344-b3fe-9de5627b0513","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57924,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/dc920a726f319e5de329044b.png"},{"id":92728019,"identity":"b3c834f4-8a2e-4c47-afb2-8f3bb53dbe34","added_by":"auto","created_at":"2025-10-03 15:01:59","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69298,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/8b24e517c5cee6f6e81d536a.png"},{"id":92728043,"identity":"2e45db02-b484-40e3-a20d-789927b21408","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7721,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/b5043e08f8168fe958c356d0.png"},{"id":92728660,"identity":"fa61ed6f-df7e-4a66-8a1e-3a73a1f1282b","added_by":"auto","created_at":"2025-10-03 15:10:01","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5572,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/7570c8bbb1499671cc489dda.png"},{"id":92728009,"identity":"0d2a63d2-8f45-470f-976e-fe581c98c963","added_by":"auto","created_at":"2025-10-03 15:01:58","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5079,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/803d22548f2c4aa4d78544ee.png"},{"id":92728665,"identity":"5a8c4cf4-f72d-4d3d-a6cf-ad2419332893","added_by":"auto","created_at":"2025-10-03 15:10:01","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39726,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/04a9ee35fc5a310136bd0a2e.png"},{"id":92728037,"identity":"ecdd14c6-d6d9-4e01-be72-e9a21d75a0c7","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12169,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/d5cb9c117b3b4547bf02f2e7.png"},{"id":92728055,"identity":"97b11fb0-e4b9-455a-ba20-aac7eec3656d","added_by":"auto","created_at":"2025-10-03 15:02:02","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12003,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/caad0927c89d345aa534d0a2.png"},{"id":92728035,"identity":"8aba145a-56db-4fde-ba16-9971ff0836b4","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10170,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/ae810b5928015d38bbb91efb.png"},{"id":92728003,"identity":"3df360f7-8622-4673-bdd4-3a3e6bfad411","added_by":"auto","created_at":"2025-10-03 15:01:57","extension":"xml","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139723,"visible":true,"origin":"","legend":"","description":"","filename":"6bbc11b636904d7a843b784d638fd95c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/a0f8eec837f2ae5644fe49c3.xml"},{"id":92728041,"identity":"197136e4-3730-4a03-b77f-de27236a78c0","added_by":"auto","created_at":"2025-10-03 15:02:01","extension":"html","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150559,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/ac0606820181c83efb100ba8.html"},{"id":92728002,"identity":"8fcdabb8-e8f9-45b3-a9a9-3ae2c8fcfd65","added_by":"auto","created_at":"2025-10-03 15:01:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87112,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of periodate oxidation of polysialic acid (PSA), and conjugation of recombinant streptokinase (SK) with oxidized PSA via reductive amination in the presence of NaCNBH\u003csub\u003e3\u003c/sub\u003e. N-acetyl neuraminic acid (Neu5Ac) units are linked via α-(2\u0026nbsp;\u0026nbsp;\u0026nbsp; 8) glycosidic bounds.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/1fc30d92ff9c13876b948f69.png"},{"id":92728027,"identity":"5f3dda64-d64f-4408-9eae-e5e4f056de4d","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":242516,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of recombinant streptokinase (SK) purification steps by 10.0% SDS-PAGE gel. Lane M: Protein marker, lane 1: Purified recombinant SK, Lane 2: Refolded recombinant SK, Lane 3: Whole cell lysate of \u003cem\u003eE. coli\u003c/em\u003e W3110 producing recombinant SK.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/dcfef91c1f3a07f618ba98ad.png"},{"id":92728658,"identity":"02cf8cff-bc37-4a53-ad86-f5a1f8ced58d","added_by":"auto","created_at":"2025-10-03 15:10:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":229446,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Influence of different molar ratios of polysialic acid (PSA) with average molecular weights of 10.0 and 39.0 kDa on the conjugation yield of PSA to streptokinase (SK). (B) Influence of incubation time on the conjugation yield of PSA to SK. (C) Influence of temperature on the conjugation yield of PSA to SK. Error bars correspond to standard deviation among three independent measurements.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/60d56cc688162cd35df1b455.png"},{"id":92728657,"identity":"4e0efa02-ab3c-4c99-aaa7-06bde07e8670","added_by":"auto","created_at":"2025-10-03 15:09:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135953,"visible":true,"origin":"","legend":"\u003cp\u003e10.0% SDS-PAGE gel analysis of streptokinase (SK) polysialylation in different molecular weights of polysialic acid (PSA). Lane M: Protein marker, Lane 1: Intact SK.; lane 2: SK polysialylated with PSA ≠ 10.0 kDa, Lane 3: SK polysialylated with PSA ≠ 39.0 kDa.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/fbb37e449fccc50bf29bbec7.png"},{"id":92728026,"identity":"c7e72fd9-e5b7-42f3-a80a-983bb1cd6806","added_by":"auto","created_at":"2025-10-03 15:02:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":115724,"visible":true,"origin":"","legend":"\u003cp\u003eElution profile of (A) native and (B) polysialylated streptokinase (SK) samples\u003cstrong\u003e \u003c/strong\u003efrom Sephadex G-100 column (30 × 0.9 cm) using 0.1 M sodium phosphate buffer (pH 7.4) with a flow rate of 0.4 mL/min on a HPLC system. (C) Molecular weight calibration curve.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/10408bb090ee5f63cee7f681.png"},{"id":92728663,"identity":"c29dd927-f5a2-4e52-bd40-d8664345c518","added_by":"auto","created_at":"2025-10-03 15:10:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92562,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of molecular weight (MW) of native and polysialylated streptokinase (SK) by Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. MALDI-TOF spectroscopy shows single peaks with a MW of: (A) 47190.0359 Da for native SK, and (B) 56505.824 Da for SK-10.0 kDa polysialic acid (PSA).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/090c3040c4395e4f92f01ebb.png"},{"id":92728007,"identity":"90cf47fd-e1a9-4085-acc8-2d0ad9a12dc8","added_by":"auto","created_at":"2025-10-03 15:01:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":196415,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Circular dichroism (CD) spectra of native and polysialylated streptokinase (SK) version in the far UV region (190-260 nm). The concentration of protein samples was 0.3 mg/mL, and the spectra were normalized to the molar elipticity. The medium buffer was 75.0 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (pH 9.0). (B) Intrinsic tryptophan fluorescence of native SK and polysialylated version. The excitation wavelength was set at 283 nm. Emission spectra were recorded from 300 to 450 nm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/f4e7f4e7ebe1de9aba9b8cb1.png"},{"id":92728656,"identity":"cad3dac6-346a-4b73-b323-c74b5a81db22","added_by":"auto","created_at":"2025-10-03 15:09:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":186094,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Hanes Woolf plot for native and polysialylated streptokinase (SK). Results are mean ± S.D. of three independent experiments, performed at 25 °C in 0.05 M sodium phosphate buffer (pH 7.4). (B) Thermal stability of native and polysialylated SK.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/6ab1fdacbd5255ed4eacb835.png"},{"id":92728008,"identity":"f701aefa-a4fc-4d00-93a2-62cc17e63084","added_by":"auto","created_at":"2025-10-03 15:01:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":14108,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetics profile of native and polysialylated streptokinase (SK) in rats. Ten rats were divided into two groups (N=5 in each group), and injected intravenously with either native SK or polysialylated variant\u003cstrong\u003e. \u003c/strong\u003eError bars correspond to standard deviation among three independent measurements.SK activity in blood plasma was measured using a colorimetric method as mentioned into the experimental procedure section..\u003cstrong\u003e \u003c/strong\u003eStatistical analysis showed significant difference (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.03 Student's \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/b00c13dcfadea328ef8ee583.png"},{"id":93050744,"identity":"cac9bf7f-dabb-456e-ab5a-032870332719","added_by":"auto","created_at":"2025-10-08 14:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2216984,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/45007ea3-de36-49a0-88a3-40ded491c8a3.pdf"},{"id":92728005,"identity":"e15686ef-d61c-4fdc-8ce3-8e77c564b281","added_by":"auto","created_at":"2025-10-03 15:01:57","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":231742,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7604522/v1/ba429c76b9be793f5d686458.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStreptokinase (SK, EC 3.4.99.0) is an enzyme produced by various strains of beta-hemolytic streptococci, and used as a thrombolytic drug for the treatment of acute myocardial infarction and pulmonary embolism.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e SK is a member of a group of medications called as fibrinolytics, and works by catalyzing the conversion of plasminogen to the active enzyme plasmin. It forms a 1:1 stoichiometric complex with plasminogen in plasma, and produces a fibrinolytic activator.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This complex hydrolyzes plasminogen molecules to generate plasmin, which subsequently degrades fibrin clots, and lyses thrombi and emboli.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e SK is in widespread clinical practice to treat acute infarction because of its function as an activator of vascular fibrinolysis.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, an important clinical problem of enzyme therapy with SK is its immunogenicity. The SK enzyme is a bacterial product, and its administration develops elevated antibody titers that cause serious allergic reactions, and neutralize drug activity. As a result, this drug should not be reused within four days of the initial dose to avoid reduced effectiveness and potential allergic reactions.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Further thrombotic events can be treated with urokinase and tissue plasminogen activator (t-PA), which are not endangered by allergic reaction and anaphylaxis. However, they are expensive, and are not accessible to broader public.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Thus, SK continues to be the primary treatment for thrombolytic therapy because of its cost-efficiency.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Given the clinical importance of this enzyme, various attempts have been done to modify SK to increase its half-life, minimize immunogenicity, and enhance its effectiveness in the body. These studies include selective removing or replacing of some amino acids, immobilization in insoluble carrier, encapsulation in nanoparticles and PEGylation.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e For example, removing 42 amino acids from C-terminal or replacing Lys59 residue with glutamic acid in SK have been reported to reduce immune response, and improve biological activity. \u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ePEGylation has been tried as a method to prolong the half-life, and decrease the immunogenic response of SK. However, this alteration hindered its effectiveness in activating plasminogen.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Hasanpour and coworkers (2021) encapsulated SK in mPEG-PLGA nanoparticles to improve its pharmacokinetic properties which resulted in prolonged half-life up to 120 minutes.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In 2020, Baharifar \u003cem\u003eet al.\u003c/em\u003e prepared PEG-grafted chitosan/SK nanoparticles, and increased biological circulation time of SK up to 120 minutes.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e In a research by Sawhney \u003cem\u003eet al.\u003c/em\u003e, a truncated PEGylated SK was designed and produced. They reported higher \u003cem\u003ein vivo\u003c/em\u003e half-life, improved clot-specificity and reduced immune-reactivity compared to the native SK.\u003csup\u003e14\u003c/sup\u003e In another work, Sawhney and other colleagues employed site-specific PEGylation, and extended circulating \u003cem\u003ein vivo\u003c/em\u003e half-life up to 20-fold compared to that of native unconjugated SK.\u003csup\u003e18\u003c/sup\u003e However, up to now, no PEGylated SK has received approval of Food and Drug Administration (FDA). In some cases, the development of antibodies against PEGylated medications has been documented, posing a significant drawback for the use of PEG.\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e So, there is a necessity to discover natural polymers that can decrease the immunogenicity of SK while maintaining its catalytic function.\u003c/p\u003e\u003cp\u003ePolysialic acid (PSA), a polymer of N-acetyl neuraminic acid, is one example of natural and biodegradable polysaccharides, which can be utilized for the conjugation of bio-therapeutics. Polysialylation technology with the use of PSA (namely colominic acid; CA) as a substitute for PEG in reducing immunogensity, and extending the circulatory half-lives of proteins has been proposed.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e.\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The rationale of this approach is that PSA creates a liquid cloud surrounding the healing molecule, and mask the immunogenic determinants, and hence increase the circulatory half-life.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In the process of conjugation, activated PSA react with lysine or N-terminus of the protein to form a stable linkage.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Polysialylated proteins exhibited enhanced stability and functionality, increased pharmacological effectiveness, reduced immunogenicity, and decreased antigenicity. The successful application of polysialylation for other bio-molecules, e.g., insulin,\u003csup\u003e27\u003c/sup\u003e catalase,\u003csup\u003e28\u003c/sup\u003e asparaginase,\u003csup\u003e29,30\u003c/sup\u003e epirubicin,\u003csup\u003e25,31\u003c/sup\u003e and uricase \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e has also been reported. This work aimed to design and develop a polysialylated variant of SK for drug delivery with reduced immunogenicity and improved half-life. For this purpose, for the first time, we chemically synthesized a bioconjugate of recombinant SK with PSA, and examined its biochemical, structural, immunological, and pharmacokinetic characteristics as compared to the native SK.\u003c/p\u003e"},{"header":"2. Experimental Procedures","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eCA (sodium salt) from \u003cem\u003eEscherichia coli\u003c/em\u003e K1 (average molecular weight (MW) 10 kDa) was procured from Fluka. CA with average MW 30.0 kDa was prepared from BIOSYNTH Ltd (United Kingdom). Sodium cyanoborohydride (NaCNBH3), \u003cem\u003eo\u003c/em\u003e-phenylenediamine (OPD), L-tryptophan (L-Trp) and 3-indoleacrylic acid (IAA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-labelled anti-rat antibodies against rat IgG and IgM were purchased from Sera-lab (Crawley Down, Sussex, UK). H-o-Val-Leu-Lys-p-nitroanilide (S-2251) was procured from Chromogenix (Italy).The pEKG-3 vector was achieved as a gift from the production department of Pasteur Institute of Iran, Karaj, Iran. Sephadex G-100 was obtained from Amersham International (Amersham, Buckinghamshire, UK). All remaining chemicals were of analytical quality.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cloning, expression and purification of recombinant SK\u003c/h2\u003e\u003cp\u003eThe cDNA sequence of \u003cem\u003eS. equisimilis\u003c/em\u003e H46A SK (accession number: X61766) was initially retrieved from Refseq of NCBI. The nucleotide sequence that encodes SK (1248 bp) was optimized for high expression in \u003cem\u003eE. coli\u003c/em\u003e strain W3110 using the Optimizer program from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genomes.urv.es/OPTIMIZER\u003c/span\u003e\u003cspan address=\"http://genomes.urv.es/OPTIMIZER\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, and produced by Biomatik Company (Canada). The optimized synthetic SK gene was amplified by polymerase chain reaction (PCR) with specific primers SKFw (5'-GA\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGAATTC\u003c/span\u003eATGATTGCTGGACCT-3') and SKRev (5'-AC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGGATCC\u003c/span\u003eTTATTTGTCGTTAGG-3'), which contained the restriction sites for \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eBam\u003c/em\u003eHI, respectively. The introduced restriction sites \u003cem\u003eEco\u003c/em\u003eRI (forward) and \u003cem\u003eBam\u003c/em\u003eHI (reverse) are underlined. For sequencing pEKG-3 plasmid, a forward sequencing primer (5'-TTGACAATTAATCATCGAAC-3') designed based on the Trp promoter region was used. The PCR product was cut with \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eBam\u003c/em\u003eHI restriction enzymes, gel purified and then ligated into the pEKG-3 expression vector previously digested with the same enzymes. The resulting recombinant construct was named pEKG-SK. The expression vector was transformed into competent cell \u003cem\u003eE. coli\u003c/em\u003e W3110, and cultured in 30 mL M9 medium containing 150 \u0026micro;g/mL L-Trp and 100 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL ampicillin under agitation at 37 \u0026ordm;C and 180 rpm for an overnight. This overnight culture was diluted 1:10 with 300 mL M9 medium containing ampicillin, but lacking L-Trp, which was grown at 37 \u0026ordm;C and 180 rpm for 1.5 h. Then, trpE promoter of recombinant gene was induced under optimized conditions by adding 20 \u0026micro;g/mL of IAA at 37 \u0026ordm;C and 180 rpm for 3 h. The cells underwent centrifugation to be gathered, then rinsed with 0.9% normal saline, resuspended in lysis buffer (consisting of 50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, and pH\u0026thinsp;=\u0026thinsp;8.0), and were sonicated on ice for 12 minutes with a 15s on and 10s off cycle with a power frequency of 9.0 kHz. The inclusion bodies (IBs) containing recombinant SK enzyme was precipitated by centrifugation (12,000 rpm for 30 minutes at 4\u0026deg;C), and washed twice with wash buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 50 mM NaCl, 10 mM EDTA, 1.0% Triton X-100 and pH 8.0,). The washed pellet was resuspended in solublization buffer (50 mM Tris\u0026ndash;HCl, 100 mM NaCl, 10 mM EDTA, 10% glycerol, 0.1 mM DTT and pH 8.0,) containing 8 M urea and incubated in 4\u0026deg;C with continuous stirring for 24 h to solubilize the IBs. Any insoluble material was removed by centrifugation at 4,000 rpm at 4\u0026deg;C for 1 h. The solublized IBs were refolded by 10-fold dilution into the buffer containing 50 mM Tris\u0026ndash;HCl, 100 mM NaCl, 10 mM EDTA, 0.1 mM DTT and pH 8.0. The refolded enzyme was applied in purification experiments.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Chromatography methods were performed on a fast performance liquid chromatography (FPLC) system (Sykam, Germany). Firstly, the sample underwent anion exchange chromatography in a DEAE\u0026ndash;Toyoperal 650 M column (3.0 cm \u0026times; 15.0 cm) utilizing 0.1 M sodium phosphate buffer (pH 7.4) with the addition of 0.1 M NaCl, and a flow rate of 0.5 mL/min. The enzyme fractions were combined, then concentrated by ultrafiltration using Amicon Ultra centrifugal filter units (10,000 MWCO, 50 mL; Millipore, Billerica, MA, USA), and applied on a Sephadex G-100 (2.5 cm \u0026times; 120 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.4).\u003csup\u003e33\u003c/sup\u003e At last, purified recombinant SK was sterilized by filtering through a 0.45 \u0026micro;m PVDF syringe filter, and the level of endotoxin was measured with LAL QCL 1000-TM Kit (LONZA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Activation of PSA\u003c/h2\u003e\u003cp\u003ePSAs with average MWs of 10.0 and 39.0 kDa were activated by oxidation of carbon 7 at the non-reducing end of the saccharide using periodate. To activate PSAs, a solution of 0.1 M sodium metaperiodate (NaIO\u003csub\u003e4\u003c/sub\u003e) was combined with PSAs (10 mg PSA/mL NaIO\u003csub\u003e4\u003c/sub\u003e) at 20\u0026deg;C, and the mixture was stirred with a magnetic stirrer for 15 minutes in the absence of light. Next, a two-fold volume of ethylene glycol was added to the reaction mixture, and was stirred for 30 minutes at 25\u0026deg;C. The oxidized PSAs were then precipitated using 70% cold ethanol, centrifuged at 15,000 rpm for 25 minutes at 20\u0026deg;C, and the resulting pellets were dissolved in a small amount of water before being stored at -20\u0026deg;C for future use.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the schematic illustration of PSA oxidation, and conjugation reaction of recombinant SK with oxidized PSA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Measurement of PSA oxidation\u003c/h2\u003e\u003cp\u003eEstimation of the PSAs oxidation was performed using 2,4 dinitrophenylhydrazine (2,4-DNPH), which forms soluble 2,4 dinitrophenyl-hydrazones when combined with carbonyl substances. For a qualitative analysis, both unoxidized and oxidized CAs were mixed with the 2,4-DNPH reagent, the mixtures were stirred, and then left to precipitate at 37\u0026deg;C until the formation of a crystalline solid was noted. A quantitative approach for measuring PSAs oxidation using 2,4 DNPH was employed using propionaldehyde as a reference. In this procedure, propionaldehyde was combined with of 2,4-DNPH, and allowed to incubate for 10 minutes at 37\u0026deg;C, after which 0.5 M NaOH was added, followed by an additional incubation at 37\u0026deg;C for 5 minutes. The resulting color intensity was assessed at 490 nm.\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Preparation of polysialylated recombinant SK\u003c/h2\u003e\u003cp\u003eRecombinant SK was chemically bonded to oxidized PSAs (10.0 and 39.0 kDa) through reductive amination with the addition of NaCNBH\u003csub\u003e3\u003c/sub\u003e at a concentration of 0.8 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To determine the best molar ratio of PSA to SK for conjugation, different ratios were mixed in 0.75 M K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (pH 9.0) with NaCNBH\u003csub\u003e3\u003c/sub\u003e (0.8 mg/mL) at 25\u0026deg;C with magnetic stirring. Samples were taken at 0, 12, 24, and 48 h, and subjected to (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e precipitation to reach 70% w/v saturation. The samples were centrifuged at 8,000 rpm at 4\u0026deg;C for 20 minutes, and the pellets containing polysialylated SK were reconstituted in 1 mL 0.15 M phosphate buffered saline (PBS; pH 7.4) buffer, and dialysed overnight at 4\u0026deg;C against 0.5 L of 0.15 M PBS buffer. The dialysates were filtered through a 0.45 mm filter to remove insoluble material, and then concentration and activity of SK were determined. Controls involved exposing the native SK to the conjugation process without PSA, according to the specified conditions. Conjugation yield was calculated according to following formula:\u003c/p\u003e\u003cp\u003eConjugation yield (%) = (activity of remaining enzyme after conjugation/activity of enzyme before conjugation) \u0026times;100 (Eq.\u0026nbsp;1)\u003c/p\u003e\u003cp\u003ePolysialylated SK was further examined, and identified using various methods as listed below.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Enzyme assay and protein determination\u003c/h2\u003e\u003cp\u003eThe amidolytic activity of both native SK and polysialylated SK was assessed with the synthetic peptide substrate S-2251. 25 \u0026micro;L of plasminogen (0.2 mg/mL) and 50 \u0026micro;L of SK sample were mixed, and incubated at 37\u0026deg;C for 10 minutes. Then, 50 \u0026micro;L of S-2251 chromogenic substrate (0.75 mg/mL) was introduced, and incubated for additional 20 minutes at 37\u0026deg;C.\u003csup\u003e12,34\u003c/sup\u003e To halt the reaction, 50 \u0026micro;L of 10% acetic acid was introduced, and the sample was examined at 405 nm using a microplate reader. One unit of SK was defined as the quantity of enzyme, which liquefies a standard clot of plasminogen at 37\u0026deg;C, and pH 7.5 for 10 minutes. Protein quantification was conducted with the Bradford technique employing Coomassie Brilliant Blue G-250.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Electrophoresis\u003c/h2\u003e\u003cp\u003eSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to detect changes in the molecular size of SK upon polysialylation. Electrophoresis was run using a 10.0% SDS gel at a current of 15 mA for 5 h. The SK and its conjugates were electrophoresed at 15 mA for 5 h. The protein bands were colored with Coomassie Brilliant Blue R-250 dye, and destained by diffusing in a solution with 40% (v/v) methanol and 10% (v/v) acetic acid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Size exclusion chromatography\u003c/h2\u003e\u003cp\u003eNative and polysialylated SKs were subjected to size exclusion chromatography in a Sephadex G-100 column (0.9 cm \u0026times; 30.0 cm) using 0.1 M sodium phosphate buffer (pH 7.4) with a flow rate of 0.4 mL/min on a HPLC system (Sykam, Germany).\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Eluent fractions were assayed for SK activity. In the column, RNA polymerase (160 kDa), lactate dehydrogenase (142 kDa), glutamate dehydrogenase (90 kDa), bovin serum albumin (66 kDa), and cytochrome c (12.4 kDa) were applied as the MW standards. The MWs for native and polysialylated variants were calculated according to the calibration curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Mass spectrometry analysis\u003c/h2\u003e\u003cp\u003eThe accurate determination of MW was determined by Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. For MALDI-TOF analysis, 1.0 mg/mL protein was desalted by passing through a G-25 Sephadex column according to the manufacturer\u0026rsquo;s instructions. The desalted protein samples were then spotted on MALDI plate, mixed with an equal volume of matrix solution of sinapinic acid in 50.0% v/v acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), air dried, and analyzed with a MALDI-TOF/TOF mass spectrometer instrument (Applied Biosystems 4800 MALDI TOF/TOF), operated in the positive ion mode. At last, the data were interpreted and processed using the instrument software (Applied Biosystems, USA).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Circular dichroism (CD)\u003c/h2\u003e\u003cp\u003eThe Aviv-215 CD spectropolarimeter was used to record the spectra in 75.0 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (pH 9.0) with protein solutions at a concentration of 0.3 mg/mL. A 1 cm cell path length was used for measuring from 190 to 260 nm (measurement parameters: bandwidth 1 mm, scanning speed 100 nm/min, response time 1.0 second). Following the correction stage which included a no-enzyme blank with 75.0 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (pH 9.0) buffer, the spectra were smoothed using the instrument software. The calculation of molar ellipticity [θM] is determined using the following formula:\u003c/p\u003e\u003cp\u003eθM\u0026thinsp;=\u0026thinsp;θobs/(c\u0026middot;d) (Eq.\u0026nbsp;2)\u003c/p\u003e\u003cp\u003eWhere θobs represents the observed ellipticity, c is the concentration of the enzyme [mol/L], and d is the path length of the quartz cuvette [cm]. At last, the wavelength was used to create a plot of θM, and the software of device was used to analyze the secondary structure content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Intrinsic fluorescence spectroscopy\u003c/h2\u003e\u003cp\u003eA Cary Eclipse spectrometer (Varian, Australia) was used to measure intrinsic Trp fluorescence emission spectra. The excitation wavelength was set at 280 nm to selectively stimulate Trp, and the emission spectra were measured from 290 to 400 nm. Samples were at a concentration of approximately 100 \u0026micro;M. The protein samples were diluted to 100 \u0026micro;M in 75.0 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e buffer (pH 9.0). For the reagent blank, the same phosphate buffer was scanned under the same conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Effect of polysialylation on kinetics parameters and thermal stability\u003c/h2\u003e\u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of native and modified SK were calculated by the Hanes-Woolf plot using GraphPad Prism 7 (GraphPad Software, La Jolla California USA) with different concentrations of plasminogen (0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 and 3.0 mg/mL). The substrate concentration divided by the reaction rate ([S]/V) was graphed against substrate concentration ([S]) with the abscissa intercept, ordinate intercept and slope being \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e and 1/\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e, respectively. For thermal stability test, the effect of temperature on the enzymatic reaction was analyzed by performing the activity test at various temperatures ranging from 20 to 50\u0026deg;C. The reaction mixture was pre-incubated at the specified temperatures for a total of 60 minutes before performing the activity assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Immunization evaluation of polysialylated SK\u003c/h2\u003e\u003cp\u003e Approval for the animal experiment was granted by the Animal Ethics Committee at Qazvin University of Medical Science under the code IR.QUMS.AEC.1402.009. Animals were executed by administrating carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) at the end of experiments. Male Wistar rats weighing 250\u0026ndash;300 g were acquired from the Pasteur Institute of Iran in Karaj. The rats were housed in a setting at 22\u0026deg;C with a relative humidity of 55%, and light/dark cycles (12 h/12 h) with food and water available throughout the study. The rats were divided into three groups (n\u0026thinsp;=\u0026thinsp;5) as follows: 1- Negative control (NC) group received 1 mL of normal saline. 2- Native group received native SK. 3-Polysialylated group received polysialylated SK. Each rat of native or polysialylated groups was given a dose of 5000.0 IU (0.2 mg/mL) of sterile solution of either native or polysialylated SK through the tail vein. Rats were immunized, and blood samples were taken on days 7, 14, and 21. The blood was then centrifuged at 4,000 rpm for 20 minutes at 25\u0026deg;C to obtain antiserum, which was stored at -30\u0026deg;C. Antibody titers in the rats were analyzed using an indirect enzyme-linked immunosorbent assay (ELISA). Polystyrene plates were covered with 100 \u0026micro;L of coating solution (50 mM sodium carbonate, pH 9.6) with 5 \u0026micro;g/mL of native or polysialylated SK in each well, then left to incubate overnight at 4\u0026deg;C. The wells were rinsed three times with 300 \u0026micro;L of PBS containing 0.1% Tween 20 (PBS-T). The unoccupied sites were blocked by adding 200 \u0026micro;L of PBS-T containing 3.0% BSA, and incubated for 2 h at 20\u0026deg;C. The wells were washed with PBS-T, and incubated with 300 \u0026micro;L of serum serially diluted in PBS-T containing 1% BSA at 20\u0026deg;C for 2 h. The wells were again washed and tap dried. Next, 100 \u0026micro;L of sheep anti-rat IgG-peroxidase conjugate (diluted to 1:750 in PBS-T with 1% BSA) was added to each well, followed by a 2 h incubation at 20\u0026deg;C. The unbound antibodies were removed by washing the wells with PBS-T, and 100 \u0026micro;L of tetramethylbenzidine (TMB) substrate solution was added. Following 30 min incubation at 25\u0026deg;C, the reaction was halted by introducing 50 \u0026micro;L of 1.0 N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e soultion, and the absorbance was measured at 450 nm. Negative control wells without SK enzyme or diluted rat serum were included in each run. The half maximal effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) was calculated according to a nonlinear regression curve fit with increasing concentrations of native or polysialylated SK.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Plasma half-life of polysialylated SK\u003c/h2\u003e\u003cp\u003eMale Wistar rats weighing between 250 and 300 g were housed in a controlled environment set at 22\u0026deg;C with 55% relative humidity, under a 12 h light/dark cycle, and provided with food and water throughout the study. Each group of rats (N\u0026thinsp;=\u0026thinsp;5) was anesthetized using ketamine and xylazine, and a warm water bag was used to induce mild vasodilation in the tail. The rats were divided into three groups (n\u0026thinsp;=\u0026thinsp;5) as follows: 1- Negative control (NC) group received 1 mL of normal saline. 2- Native group received native SK. 3-Polysialylated group received polysialylated SK. Approximately 7500.0 IU (0.34 mg/mL) of either native SK or polysialylated variant in sterile solution was administered to rats through the tail vein. Around 50 \u0026micro;l of complete blood samples were taken from rats through tail transection at 10, 20, 30, 60, 120, 180, and 360 minutes following drug administration. The blood samples were put into heparinized Eppendorf tubes and centrifuged at 3,000 rpm for 10 minutes at 25\u0026deg;C to separate plasma, then stored at -20\u0026deg;C. SK activity in plasma was measured using a colorimetric method as mentioned before.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll tests were carried out at least three times, and each value represented the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software, La Jolla California USA) to compare the statistical significance between the native and polysialylated variants. Differences with \u003cem\u003ep\u003c/em\u003e-values below 0.05, and confidence levels of 95.0% were considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Production of recombinant SK enzyme\u003c/h2\u003e\u003cp\u003eFor bacterial production of SK enzyme, a recombinant plasmid based on the expression vector pEKG-3 was constructed. The synthetic gene of SK was assembled based on the \u003cem\u003eE. coli\u003c/em\u003e W3110 codon usage. PCR amplification of codon-optimized SK generated a 1248 bp fragment. The obtained PCR product was cut with \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eBam\u003c/em\u003eHI restriction enzymes, purified from the gel, and ligated into pEKG-3 vector. The recombinant plasmid was confirmed by restriction analysis, and DNA sequencing, and then transferred into \u003cem\u003eE. coli\u003c/em\u003e W3110 for expression. The purified refolded recombinant SK enzyme was duly detected by SDS-PAGE. The SK migrated at approximately 47.0 kD by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The yield of enzyme production and biomass concentration under the optimized conditions were estimated to be 19.0% of total protein, and 17.5 g per 1 L culture medium, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Synthesis and characterization of recombinant polysialylated SK\u003c/h2\u003e\u003cp\u003eTo obtain the optimal condition for enzyme polysialylation, CAs (10.0 and 39.0 kDa) were coupled to SK at 50:1, 100:1, 150:1, and 200:1 PSA: SK molar ratios. The reactions were followed by ammonium sulfate precipitation, and conjugation yields were calculated based on the PSAs found in the precipitate (Fig.\u0026nbsp;3A). As seen, the highest conjugation yield (64.52%) was achieved by 10.0 kDa PSA at molar ratio of 200:1. Thereafter, the influence of incubation time (Fig.\u0026nbsp;3B) and temperature (Fig.\u0026nbsp;3C) on the conjugation yield was also investigated. The results showed that 24 h and 25\u0026deg;C were the best time, and temperature for conjugation reaction, respectively. The purified native SK migrated at approximately 47.0 kD by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). When SDS-PAGE was applied to detect changes in the MW of SK, polysialylated SKs exhibited different electrophoresis migration at around 55.0 kDa and 80.0 kDa, which confirmed the conjugation of SK to 10.0 and 30.0 kDa PSAs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, according to the low conjugation yield (21.23%), faint band of SK-39.0 kDa PSA variant compared to the SK-10.0 kDa PSA at equivalent protein concentrations in SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as well as significant loss of activity of SK-39.0 kDa PSA (results not shown), we decided to do the next experiments only with SK-10.0 kDa PSA variant. The samples were further analyzed using size exclusion chromatography in a Sephadex G-100 column (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As observed, a peak of native SK was detected at approximately 12 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas polysialylated SK eluted at approximately after approximately 22 minutes of injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) indicating the higher MW of the polysialylated variant. The exact MWs of native SK and SK-10.0 kDa PSA were determined by MALDI-TOF mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The data showed an exact MW of 56.5 kDa for SK-10.0 kDa PSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) which nearly matches the calculated value by SDS-PAGE. The monodisperse composition was confirmed for both samples (just one peak) with no detectable shoulders or minor peaks related to impurities or truncated forms. Conjugation reaction was also analyzed by CD and fluorescence spectroscopy methods. CD spectra showed that the polysialylation induces a decrease in the far UV CD signal, suggesting an increase in the protein alpha helix content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The intrinsic Trp fluorescence intensity of polysialylated SK increased compared to the native version (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating changes in the microenvironments of Trp residues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Kinetic parameters and thermal stability\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of polysialylation on the enzyme\u0026rsquo;s kinetics, kinetics data were determined based on the initial velocity measurements of the S-2251 degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). In the present study, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values estimated by the Hanes Woolf plot for native and polysialylated SKs were 31.1 and, 32.5 mg/mL, respectively. From the same plot, calculated \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values for native and polysialylated enzymes were 283.5, and 294.2 \u0026micro;mol min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The thermal stability of polysialylated variant was also studied. As observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), polysialylated SK retained its activity up to 91.0% at 55.0\u0026deg;C while native enzyme lost nearly 50.0% of original activity at this temperature.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.4. Evaluation of immunization and pharmacokinetic of polysialylated SK\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eResults (total IgG) in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that, at equivalent protein concentrations, polysialylated SK elicited nearly 62.0% lower antibody production compared to the native variant. Moreover, the lower value (16000.0 IU\u0026thinsp;\u0026plusmn;\u0026thinsp;65.0) of SK-10.0 kDa PSA concentration (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) that achieved EC\u003csub\u003e50\u003c/sub\u003e exhibited that polysialylated SK was more potent compared to the native enzyme. The \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics of native SK and polysialylated variant were also assessed. The enzyme activity was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.03) higher in the rats injected with polysialylated SK than in those injected with native enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The comparison of pharmacokinetics parameters estimated by statistical analysis is also depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Polysialylation of SK yielded preparation with a longer plasma half-life (2.21 h) compared to the native variant (0.5 h).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of polysialylation on IgG titer, and half-maximal effective concentration (EC\u003csub\u003e50\u003c/sub\u003e). Antisera raised against native and polysialylated streptokinase (SK). The significant difference between each of the coatings with polysialylated SK to the coating with native enzyme was \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTotal IgG titer (OD) (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003cp\u003e7 Days 14 Days 21 Days\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (IU)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e320\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1120\u0026thinsp;\u0026plusmn;\u0026thinsp;45.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1750\u0026thinsp;\u0026plusmn;\u0026thinsp;55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26000.0 IU\u0026thinsp;\u0026plusmn;\u0026thinsp;50.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePolysialylated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e420\u0026thinsp;\u0026plusmn;\u0026thinsp;35.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e690\u0026thinsp;\u0026plusmn;\u0026thinsp;42.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16000.0 IU\u0026thinsp;\u0026plusmn;\u0026thinsp;65.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eAll values are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three measurements.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of pharmacokinetics parameters of native streptokinase (SK) and polysialylated variant.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSK variant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHalf-Life (h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVolume of distribution (L/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClearance (L/hr/kg)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5 \u0026plusmn; 0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePolysialylated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.46\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003eThe values represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of two independent experiments.\u003c/p\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.02: significant difference vs. native variant.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePolymer-based drug delivery systems have been utilized in biomedical applications in attempts to improve the therapeutic activity of drug materials. Potential advantages of these delivery mechanisms include an increased or prolonged duration of pharmacologic activity, a decrease in adverse effects, increased patient compliance and quality of life.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Among the available techniques, polymer\u0026ndash;drug conjugates have exhibited clinical and commercial success in the field of drug delivery. Polymer-drug conjugates are a part of therapeutics, in which the therapeutic agent is chemically linked to a polymer instead of being encapsulated into it. They offer distinct features, such as reduced drug toxicity, increased drug solubility, enhanced drug bioavailability, biocompatibility, reduced immunogenicity, and improved pharmacokinetic parameters. Such strategies have the potential to develop as the next generation protein therapeutics. Over the past decade, numerous polymer\u0026ndash;drug conjugates-based formulations entered the market, and several others are in clinical trials.\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e PEG is among the most well-understood and utilized synthetic polymers, and has been FDA approved as a material for polymer\u0026ndash;drug conjugates. Grafting of hydrophilic macromolecules such as PEG onto the surface of drugs has proved successful in prolonging presence in blood circulation, and reducing immunogenicity with consequent increase of therapeutic efficacy. \u003csup\u003e14,18\u003c/sup\u003e However, conjugation of PEG to enzymes often leads to substantial reduction of their activity. Moreover, the non-biodegradable PEG is expected to accumulate in the lysosomes following endocytosis of the conjugates, possibly leading to toxicity on chronic use. Despite various successes of PEGylation, some products were withdrawn from the market because of polymer toxicity or limited drug loading. To overcome the major issues of biodegradability and toxicity, natural polymers, such as PSA is being explored as a safe and alternative solution.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Polysialylation with the use of the highly hydrophilic and biodegradable polymer of PSA as an alternative to PEG in prolonging the circulatory half-lives of proteins has been suggested. PSA can be used to protect therapeutic molecules from the host immune system, and improve their pharmacokinetics.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSK is a highly effective thrombolytic agent, and often used in less affluent societies for the myocardial infarction.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e However, if some of its major drawbacks can be rectified, it has the potential to occupy centre-stage as an effective thrombolytic, especially for ischemic strokes. \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e Hence, several studies have tried to increase half-life, minimize immunogenicity and improve the biological efficacy with different mechanisms, such as protein engineering and developing delivery systems for SK.\u003csup\u003e8,16,17\u003c/sup\u003e It has been shown that PEGylation of SK considerably enhances its therapeutic attributes.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e The production of antibodies against PEGylated drugs has also been reported in some cases, and this proves to be a great disadvantage for PEG applications.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e So, there is a need to find a biocompatible polymer for conjugation to SK that can reduce the immunogenicity without compromising its enzymatic activity.\u003c/p\u003e\u003cp\u003eIn the present investigation, polysialylation of SK was attempted through \u003cem\u003ein vitro\u003c/em\u003e chemical conjugation. For bacterial production of SK enzyme, a recombinant plasmid based on the generic expression vector pEKG-3 was constructed. The recombinant expressed SK migrated at approximately 47.0 kD by SDS-PAGE. In polysialylation of enzyme, the highest conjugation yield (64.52%) was achieved by 10.0 kDa PSA and 200:1 molar ratio. The results showed that 24 h and 25\u0026deg;C were the best time and temperature for conjugation reaction, respectively. Initial evidence that SK was covalently linked to PSA to form a sialyated enzyme was obtained by SDS-PAGE. The polysialyated SKs exhibited different electrophoresis migrations at around 55.0 kDa and 80.0 kDa which confirmed the conjugation of SK to PSA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, according to the low conjugation yield (21.23%), faint band of SK-39.0 kDa PSA variant compared to the SK-10.0 kDa PSA at equivalent protein concentrations in SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as well as significant loss of activity of SK-39.0 kDa PSA (results not shown), we decided to do the next experiments only with SK-10.0 kDa PSA variant. It seems that PSA with higher MW such as 30.0 kDa is more suitable for the small protein or peptides. This is supported by work on insulin polysialylation in which 39.0 kDa PSA was optimized for conjugation.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In contrast, for the several other proteins, e.g., asparaginase,\u003csup\u003e30\u003c/sup\u003e catalase,\u003csup\u003e28\u003c/sup\u003e and uricase,\u003csup\u003e32\u003c/sup\u003e 10.0 kDa PSA has been utilized. The exact MWs of native SK and SK-10.0 kDa PSA were determined by MALDI-TOF mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Conjugation reaction was also analyzed by CD and fluorescence spectroscopy. CD spectra showed that polysialyation induces a decrease in the far UV CD signal which suggests an increase in the protein alpha helix content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This finding implies that polysialyated SK has greater protein stability than the native version. Structural stability and conformational state of polysialylated enzyme was further analyzed by measuring the native intrinsic Trp fluorescence emission at 290 nm, while being excited from 290 to 400 nm. Intrinsic protein fluorescence deriving from the naturally fluorescent Trp can provide information on the conformational changes of proteins, and their interactions with other molecules.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e The intrinsic Trp fluorescence intensity of polysialylated SK increased compared to the native version (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating changes in the microenvironments of Trp residues. As a general rule, in the folded state, Trp residues are mainly located in the hydrophobic core of proteins. When they become exposed to solvents (a hydrophilic environment) in an unfolded state, they will have reduced fluorescence intensity.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Thus, it can be said that the increment of fluorescence intensity in polysialylated enzyme means that stability of SK was increased by immobilizing on PSA polymer, consistent with the CD results. On the other hand, there was no notable difference in the maximum fluorescence emission wavelength (λmax) between native and polysialylated SK, i.e., no unfavorable conformational changes in SK polysialylation are occurred. The interpretation of the fluorescence spectroscopy leads to the conclusion that polysialylation of SK not only changes the conformational state, but also stabilizes the enzyme structure. Chemical modification of enzymes with polymers is known to often adversely affect their kinetic parameters, and thus limit their efficacy.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Evidently, a prime consideration in employing polysialylation for enzymes is the maintenance of their function. In the present study, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values estimated by the Hanes Woolf plot for native and polysialylated SKs were 31.1 and, 32.5 mg/mL, respectively. From the same plot, calculated \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e values for native and polysialylated enzymes were 283.5, and 294.2 \u0026micro;mol min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. As can be found, \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of polysialylated SK is slightly higher than that of native SK, showing that the attached PSA molecule does not significantly affect the enzyme affinity toward its substrate. It is likely that this phenomenon was due to the little conformational change of SK on PSA attachment, rather than to the diffusional barrier of PSA. In other words, it means that, PSA polymer had not significant structural influence on the active site of SK. This result might also be attributed to the limited number of PSAs bound per molecule of enzyme. Taken together, the kinetic results indicate that the covalent coupling of PSA to SK does not affect its substrate specificity, and the polysialylated enzyme will be able to effectively act \u003cem\u003ein vitro.\u003c/em\u003e Similar results have been obtained for the polysialylation of other enzymes, e.g., catalase,\u003csup\u003e28\u003c/sup\u003e uricase \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and asparginase. \u003csup\u003e30\u003c/sup\u003e The thermal stability of polysialylated variant was also studied. As observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), polysialylated SK retained its activity up to 91.0% at 55.0\u0026deg;C while native enzyme lost nearly 50.0% of original activity at this temperature. The higher activity of polysialylated SK at temperature of 55\u0026deg;C after 1 h of incubation suggests that polysialylation has improved the overall stability of the enzyme. Indeed, this is an interesting advantageous for any pharmaceutical application.\u003c/p\u003e\u003cp\u003eUnwanted immunogenicity of conjugated proteins to polymers (e.g., PEG or PSA) can cause undesirable side effects, and render them ineffective. The immune-reactivity of polysialylated SK and native variant was measured by indirect ELISA using antisera raised against each of the antigen preparations. Results (total IgG) indicate that, at equivalent protein concentrations, polysialylated SK elicited nearly 62.0% lower antibody production compared to the native variant. Our interpretation of these findings is that the conjugated PSA chains sterically hinder the binding of IgG molecules to the relevant antigenic sites on SK, leading in turn to reduce antigenicity. In other words, PSA chains may help to mask some antigenic determinants (epitopes), and prevent complexing of SK with its antibodies. This was in line with the finding that immune complexes are formed by spatial complementarity, and that the forces that bind antigen and antibody together are weakened by an increased distance between the two molecules.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Another reason that may have contributed to the reduction of antigen\u0026ndash;antibody binding would be the generation of repulsive forces due to the negative charge of the enzyme-bound PSAs. Moreover, it is conceivable that loss of some of the free-amino groups of SK on polysialylation has rendered the modified enzyme intrinsically more negatively charged. In accordance with our results, similar reductions in immunogenicity with polysialylation have been reported for asparginase,\u003csup\u003e29\u003c/sup\u003e insulin,\u003csup\u003e27\u003c/sup\u003e erythropoietin,\u003csup\u003e42\u003c/sup\u003e uricase, \u003csup\u003e32\u003c/sup\u003e and catalase.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics of native SK and polysialylated variant were also assessed. The enzyme activity was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.03) higher in the rats injected with polysialylated SK than in those injected with native enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The comparison of pharmacokinetics parameters estimated by statistical analysis is also depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Polysialylation of SK yielded preparation with a longer plasma half-life (2.21 h) compared to the native variant (0.5 h). In comparison, half-life of polysialylated SK is longer than the reported values for encapsulated SK in mPEG-PLGA (2.0 h),\u003csup\u003e13\u003c/sup\u003e PEG-grafted chitosan/SK nanoparticles (2.0 h),\u003csup\u003e12\u003c/sup\u003e and truncated PEGylated SK (2.0 h).\u003csup\u003e14\u003c/sup\u003e Indeed, the increased period of pharmacological activity of polysialylated SK suggests that polysialylation contributes to longer retention of enzyme activity. The similar results with catalase,\u003csup\u003e28\u003c/sup\u003e insulin,\u003csup\u003e27\u003c/sup\u003e asparaginase,\u003csup\u003e29\u003c/sup\u003e interferon α-2b \u003csup\u003e23\u003c/sup\u003e and several other proteins strongly show that the presence of PSA chains on the molecules allows retention of much the activity in polysialylated therapeutics.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summarize, the data presented in this paper reveals that the enhanced properties obtained by SK-10.0 kDa PSA, particularly structural stability, reduced immunogenicity, and extended half-life can contribute to ameliorate the therapeutic value of this thrombolytic drug. It is noteworthy that polysialylation technology offers a promising strategy for the synthesis of a novel version of SK with improved pharmacological properties. In the meanwhile, since this research is our preliminary data, further studies is required to confirm the efficacy of this formulation on human. In light of this, we plan to continue our work with further optimization of conjugation reaction, additional preclinical studies, and human clinical trials of polysialylated SK.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHamid Shahbazmohammadi\u003c/strong\u003e\u0026minus; \u003cem\u003eCellular and Molecular Research Center, Research Institute for Prevention of Non-Communicable Diseases,\u0026nbsp;\u003c/em\u003e\u003cem\u003eQazvin University of Medical Sciences\u003c/em\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cem\u003eQazvin, Iran\u003c/em\u003e;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehttps://orcid.org/0000-0002-2099-1812;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEmail: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEskandar Omidinia\u003c/strong\u003e \u0026minus;\u0026nbsp;\u003cem\u003eEnzyme Technology Laboratory, Department of Biochemistry, Genetic and Metabolism, Research Group, Pasteur Institute of Iran, Tehran, Iran\u003cstrong\u003e;\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ehttps://orcid.org/0000-0002-3002-5714\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHamid Shahbazmohammadi:\u003c/strong\u003e Writing\u0026ndash;review \u0026amp; editing, Conceptualization, Methodology, Formal analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEskandar Omidinia\u003c/strong\u003e: Supervision, Project administration, Funding acquisition. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Qazvin University of Medical Sciences (project number: 402000043).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKiran GR, Chandrasekhar P, Mohammad Ali S. Clinical outcomes of patients with mitral prosthetic valve obstructive thrombosis treated with streptokinase or tenecteplase. Indian Heart J. 2021;73:365\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabbal A, Mohanty S, Khasa YP. Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of \u003cem\u003ePichia pastoris\u003c/em\u003e: a case study with proteolysis prone streptokinase. Int J Biol Macromol. 2021;180:760\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLocke M, Rigsby P, Longstaff C. An international collaborative study to establish the WHO 4th international standard for streptokinase: communication from the SSC of the ISTH. J Thromb Haemost. 2020;181:501\u0026ndash;1505.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyinuola YA, Brito-Robinson T, Ayinuola O, Beck JE, Cruz-Topete D, Lee SW, Ploplis VA, Castellino FJ. Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence. J Biol Chem. 2021;296:100099.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahmoud AAA, Mahmoud HE, Mahran MA, Khaled M. Streptokinase versus unfractionated heparin nebulization in patients with severe acute respiratory distress syndrome (ARDS): a randomized controlled trial with observational controls. J Cardiothorac Vasc Anesth. 2020;34:436\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Dabaa E, Okasha H, Samir S, Nasr SM, El-Kalamawy HA, Saber MA. Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity. Arab J Chem. 2022;15:103799.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDey S, Guchhait KC, Manna T, Panda AK, Patra A, Mondal SK, Ghosh C. Evolutionary and compositional analysis of streptokinase including its interaction with plasminogen: an in silico approach. Gene Rep. 2022;29:101689.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaheri MN, Behzad-Behbahani A, Rafiei Dehbidi G, Salehi S, Sharifzadeh S. Engineering, expression and purification of a chimeric fibrin-specific streptokinase. Protein Expression Purif. 2016;128:14\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehrad H, Foletti A. Effect of optison microbubbles-mediated B-mode ultrasound- guided focused low-level confocal dual pulse electrohydraulic shock wave-assisted streptokinase thrombolysis on embolic carotid artery. Atherosclerosis. 2021;331:e263.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulpuri VB, Vaswani P, Gupta R, Rana SS, Kang M, Gorsi U, Gupta R. Fr298 streptokinase has a role in salvaging patients undergoing step up treatment who fail large volume saline lavage. Gastroenterol. 2021;160:S\u0026ndash;289.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhargava V, Gupta R, Vaswani P, Jha B, Rana SS, Gorsi U, Kang M, Gupta R. Streptokinase irrigation through a percutaneous catheter helps decrease the need for necrosectomy and reduces mortality in necrotizing pancreatitis as part of a step-up approach. Surgery. 2021;170:1532\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaharifar H, Khoobi M, Arbabi Bidgoli S, Amani A. Preparation of PEG-grafted chitosan/streptokinase nanoparticles to improve biological half-life and reduce immunogenicity of the enzyme. Int J Biol Macromol. 2020;143:181\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHasanpour A, Esmaeili F, Hosseini H, Amani A. Use of mPEG-PLGA nanoparticles to improve bioactivity and hemocompatibility of streptokinase: in-vitro and in-vivo studies. Mater Sci Eng C. 2021;118:111427.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSawhney P, Katare K, Sahni G. PEGylation of truncated streptokinase leads to formulation of a useful drug with ameliorated attributes. PLoS ONE. 2016;11:e0155831.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehrad H, Shokouhi MF. Sonothrombolysis of embolic carotid artery, using pesda microbubbles- mediated B-mode ultrasound- guided extracorporeal pulsed low- level focused confocal dual frequency ultrasound accompanied by streptokinase administration. Atherosclerosis. 2021;331:e264.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdivitiya, Khasa YP. The evolution of recombinant thrombolytics: current status and future directions. Bioengineered. 2017;8:331\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSevostyanov MA, Baikin AS, Sergienko KV, Shatova LA, Kirsankin AA, Baymler IV, Shkirin AV, Gudkov SV. Biodegradable stent coatings on the basis of PLGA polymers of different molecular mass, sustaining a steady release of the thrombolityc enzyme streptokinase. React Funct Polym. 2020;150:104550.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSawhney P, Kumar S, Maheshwari N, Guleria SS, Dhar N, Kashyap R, Sahni G. Site-specific thiol-mediated PEGylation of streptokinase leads to improved properties with clinical potential. Curr Pharm Des. 2016;22:5868\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTorr\u0026eacute;ns I, Ojalvo AG, Seralena A, Pupo E, Lugo V, P\u0026aacute;ez R. A Mutant streptokinase lacking the C-terminal 42 amino acids is less reactive with preexisting antibodies in patient sera. Biochem Biophys Res Commun. 1999;266:230\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePelingon R, Pegg CL, Zacchi LF, Phung TK, Howard CB, Xu P, Hardy MP, Owczarek CM, Schulz BL. Glycoproteomic measurement of site-specific polysialylation. Anal Biochem. 2020;596:113625.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNajjari A, Shahbazmohammadi H, Nojoumi SA, Omidinia E. PASylated urate oxidase enzyme: enhancing biocatalytic activity, physicochemical properties, and plasma half-life. ACS Omega. 2022;7:46118\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConstantinou A, Chen C, Deonarain MP. Polysialic acid and polysialylation to modulate antibody pharmacokinetics, in therapeutic proteins. Wiley Online Libaray; 2012. pp. 95\u0026ndash;115.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGregoriadis G, Jain S, Papaioannou I, Laing P. Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Int J Pharm. 2005;300:125\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLindhout T, Iqbal U, Willis LM, Reid AN, Li J, Liu X, Moreno M, Wakarchuk WW. Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 2011, 108, 7397\u0026ndash;7402.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang T, Zhou S, Hu L, Peng B, Liu Y, Luo X, Liu X, Song Y, Deng Y. Polysialic acid-polyethylene glycol conjugate-modified liposomes as a targeted drug delivery system for epirubicin to enhance anticancer efficiency. Drug Deliv Transl Res. 2018;8:602\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmirnov IV, Vorobiev II, Belogurov AA, Genkin DD, Deyev SM, Gabibov AG. Chemical polysialylation of recombinant human proteins, in glyco-engineering: methods and protocols, Castilho A. Editor. 2015, Springer, New York, 2015, pp. 389\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJain S, Hreczuk-Hirst DH, McCormack B, Mital M, Epenetos A, Laing P, Gregoriadis G. Polysialylated insulin: synthesis, characterization and biological activity in vivo. Biochim Biophys Acta-Gen Subj. 2003;1622:42\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandes AI, Gregoriadis G. Synthesis, characterization and properties of sialylated catalase, Biochim. Biophys. Acta. Protein Struct Mol Enzymol. 1996;1293:90\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandes AI, Gregoriadis G. The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics. Int J Pharm. 2001;217:215\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandes AI, Gregoriadis G. Polysialylated asparaginase: preparation, activity and pharmacokinetics. Biochim Biophys Acta Protein Struct Mol Enzymol. 1997;1341:26\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang T, Zhou S, Hu L, Peng B, Liu Y, Luo X, Song Y, Liu X, Deng Y. Polysialic acid-modifying liposomes for efficient delivery of epirubicin, in-vitro characterization and in-vivo evaluation. Int J Pharm. 2016;515:449\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePunnappuzha A, PonnanEttiyappan JB, Sekhar Nishith R, Hadigal S, Ganapathi Pai P. Synthesis and characterization of polysialic ccid\u0026ndash;uricase conjugates for the treatment of hyperuricemia. Int J Pept Res Ther. 2014;20:465\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShahbazmohammadi H, Omidinia E. Development and application of aqueous two-phase partition for the recovery and separation of recombinant phenylalanine dehydrogenase. Iran J Chem Chem Eng. 2008;27:119\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShahbazmohammadi H, Omidinia E, Sahebghadam Lotfi A, Saghiri R. Preliminary report of NAD\u003csup\u003e+\u003c/sup\u003e-dependent amino acid dehydrogenase producing bacteria isolated from soil. Iranianian Biomed J. 2007;11:131\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShahbazmohammadi H, Omidinia E, Dinarvand R, Taherkhani HA. Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase. Bioprocess Biosyst Eng. 2010;33:317\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlven S, Nqoro X, Buyana B, Aderibigbe BA. Polymer-drug conjugate, a potential therapeutic to combat breast and lung cancer. Pharmaceutics. 2020;12:406.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThakor P, Bhavana V, Sharma R, Srivastava S, Singh SB, Mehra NK. Polymer\u0026ndash;drug conjugates: recent advances and future perspectives. Drug Discov Today. 2020;25:1718\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J Pharm Sci. 2010;99:2557\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEkladious I, Colson YL, Grinstaff MW. Polymer\u0026ndash;drug conjugate therapeutics: advances, insights and prospects. Nat Rev Drug Discovery. 2019;18:273\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLarson N, Ghandehari H. Polymeric conjugates for drug delivery. Chem Mater. 2012;24:840\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu J, Lu S, Zheng Z, Zhu L, Zhan X. Modification with polysialic acid\u0026ndash;PEG copolymer as a new method for improving the therapeutic efficacy of proteins. Prep Biochem Biotechnol. 2016;46:788\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeng H, Jain S, Lockshin C, Shaligram U, Martinez J, Genkin D, Hill DB, Ehre C, Clark D, Hoppe. H.Clinical application of polysialylated deoxyribonuclease and erythropoietin. Recent Pat Drug Deliv Formul. 2018;12:212\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlanazi AZ, Alhazzani K, Mostafa AM, Barker J, El-Wekil MM, Ali A. M.B.H. Highly selective fluorometric detection of streptokinase via fibrinolytic release of photoluminescent carbon dots integrated into fibrin clot network. Microchem J. 2024;197:109800.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurkia HB, Souissi S, Chabaane F, Chermiti I, Fadhel R, Keskes S, Bekir A, Ghazali H. 172 Fibrinolysis of ST elevation myocardial infarction in emergency department: prognosis of elderly patients treated with streptokinase. Resuscitation. 2024;203:S88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003edos Rodrigues S, Delgado FH, Santana da Costa GG, Tasic T. Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts. BBA Adv. 2023;3:100091.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGooran N, Kopra K. Fluorescence-based protein stability monitoring-a review. Int J Mol Sci. 2024;25:1764.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao Y, Zhao J. Innovative application of Coomassie Brilliant Blue: a simple, economical, and effective determination of water-insoluble protein surface hydrophobicity. Anal Methods. 2015;8:790\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDelves PJ, Martin SJ, Burton DR, Roitt IM. Roitts\u0026rsquo;s Essential Immunology. 8th ed. Oxford, UK: Blackwell Scientific; 1994. pp. 81\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Conjugation, Half-life, Immunogenicity, Polysialic acid (PSA), Streptokinase (SK)","lastPublishedDoi":"10.21203/rs.3.rs-7604522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7604522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStreptokinase (SK, EC 3.4.99.0) is an enzyme produced by beta-haemolytic \u003cem\u003estreptococcus\u003c/em\u003e, and catalyzes the conversion of plasminogen to its active and proteolytic form, plasmin. SK is used in medicine as a fibrinolytic enzyme system for dissolving clots in conditions such as heart attacks, ling artery emboli, vein thrombosis, and occlusions of arteries. Despite the impressive use of this thrombolytic drug, its immunogenicity and short biological half-life are major challenges that limit its efficacy in clinical setting. In this communication, we studied the polysialylation of SK with the aim to improve the pharmacokinetics of this drug in medicine. The skc-2 gene from \u003cem\u003eS. equisimilis\u003c/em\u003e ATCC 9542 was codon optimized, and produced as a recombinant enzyme in \u003cem\u003eEscherichia coli\u003c/em\u003e W3110. Recombinant SK was covalently conjugated to oxidized polysialic acid (PSA; also referred to as colominic acid; CA) via reductive amination in the presence of NaCNBH\u003csub\u003e3\u003c/sub\u003e. The native and polysialylated variants were compared in terms of structural properties, enzyme kinetics, stability, immunization and biological half-life. The best molecular weight of PSA, optimum molar ratio, incubation time, and temperature for conjugation reaction of PSA to SK were determined to be 10.0 kDa, 200: 1, 24 h and 25\u0026deg;C, respectively. SDS-PAGE analysis revealed a band at 55.0 kDa for conjugated SK which confirmed the conjugation of PSA to SK. The exact molecular weight of SK-10.0 kDa PSA was determined to be 56.5 kDa by MALDI-TOF spectrometry mass which matches the calculated value by SDS-PAGE. Polysialylation induced a decrease in the far UV CD signal, suggesting an increase in the protein alpha helix content. The intrinsic fluorescence intensity of polysialylated SK increased compared to the native version, meaning that stability of SK was increased by immobilizing on PSA polymer, consistent with the CD results. \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of polysialylated SK was slightly higher than that of native SK, which showed that the attached PSA molecules to the enzyme did not significantly reduce the substrate specificity. Polysialylated SK elicited nearly 63.0% lower antibody production compared to the native variant. Native and polysialylated SKs exhibited plasma half-life of 0.5 and 2.21 h, respectively, implying that the modified variant has a 4.42-fold longer residence time in body. Briefly, comparative studies with native and PSA-conjugated enzymes show that polysialylation can be useful in enhancing the therapeutic efficacy of SK. It is worth emphasizing that this is the first report describing the use of polysialylation technology to improve the pharmaceutical properties of SK.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Synthesis and Analysis of Biochemical and Pharmaceutical Properties of Polysialylated Recombinant Streptokinase Enzyme","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 15:01:46","doi":"10.21203/rs.3.rs-7604522/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b2942aa-80e6-4a48-bc46-a225030e3f31","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-08T14:09:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 15:01:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7604522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7604522","identity":"rs-7604522","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}