Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features

Title:Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features

Volume: 24 Issue: 10

Author(s): Narges Norouzzadeh Alinodehi, Hamideh Behrooz, Milad Sabaei, Farahnaz Khoshdel Nezamiha and Reza Arabi Mianroodi*

Affiliation:

• R&D Department, Research and production complex, Pasteur Institute of Iran, Tehran, Iran

Keywords: Streptokinase, plasminogen, site directed mutagenesis, cysteine, activity, docking.

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.

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.

Cite this article as:

Alinodehi Norouzzadeh Narges, Behrooz Hamideh, Sabaei Milad, Nezamiha Khoshdel Farahnaz and Mianroodi Arabi Reza*, Multiple Mutations on α, β and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features, Current Pharmaceutical Biotechnology 2023; 24 (10) . https://dx.doi.org/10.2174/1389201024666221124151623