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 (SK45-319cys, SK263-319cys, and SK45-263-319cys) 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, SK263-319cys 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.
