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Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study

[ Vol. 20 , Issue. 1 ]

Author(s):

Narges N. Alinodehi, Sanaz Sadeh, Farahnaz K. Nezamiha, Malihe Keramati, Mehdi Hasanzadeh and Reza A. Mianroodi*   Pages 76 - 83 ( 8 )

Abstract:


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.

Keywords:

Streptokinase, mutation, cysteine substitution, cloning, activity assay, expression.

Affiliation:

R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, R&D Department, Research and Production Complex, Pasteur Institute of Iran, Karaj



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