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Title

POINT MUTATION IN AMINO ACID 263 OF STREPTOKINASE GENE AS WELL AS CLONING ANDEXPRESSION OF THE CYSTEINE CONTAINING MUTATED PROTEIN

Pages

 Start Page 249 | End Page 257

Abstract

 Streptokinase is one of the best known THROMBOLYTIC agents with widespread clinical use. However, its use isnot risk-free due to its immunogenicity, hemorrhagic complications and relatively short half-life in circulation. Specific PEGYLATION of cysteine residue is a useful technique for reducing most of these complications. The aim of this study was designing and producing a cysteine containing mutant of streptokinase, to be used for specific PEGYLATION. Glut -amic acid 263, which is a surface amino acid in the structure of streptokinase protein, was selected for replacement with cysteine amino acid by site directed mutagenesis. The Glu263 codon was changed to cysteine codon by SOEing PCR technique. Then, the intact and mutated streptokinase genes were inserted into expression vector pET-26b (+). The constructs were transformed to Escherichia.coli Rosetta (DE3) strain and the proteins were expressed by IPTG induction.The proteins were confirmed by SDS-PAGE and western blot analysis, purified by Ni-NTA agarose affinity chromatogram phyunder denaturing condition with urea and Sephadex G-25 column was applied to remove urea to refold the proteins.This study indicated that by using aforesaid vector and host, cysteine containing mutant gene is expressed well and it will be appropriate for specific PEGYLATION.

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