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Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features.
- Source :
-
Current pharmaceutical biotechnology [Curr Pharm Biotechnol] 2023; Vol. 24 (10), pp. 1326-1334. - Publication Year :
- 2023
-
Abstract
- 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.<br />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.<br />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 <subscript>45-319cys</subscript> , SK <subscript>263-319cys</subscript> , and SK <subscript>45-263-319cys</subscript> ) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate.<br />Results: Based on the kinetic results, SK <subscript>263-319cys</subscript> 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.<br />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.<br /> (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
Details
- Language :
- English
- ISSN :
- 1873-4316
- Volume :
- 24
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- Current pharmaceutical biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 36424778
- Full Text :
- https://doi.org/10.2174/1389201024666221124151623