Search

Background: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function., Objectives: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation., Methods: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK 45-319cys , SK 263-319cys , and SK 45-263-319cys ) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate., Results: Based on the kinetic results, SK 263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with μ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference., Conclusion: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment., Objective: This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation., Method: In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed., Results: According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity., Conclusion: SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)