The main objective of the present study is cloning streptokinase gene of mutant strain (UV and ethyl methyl sulphonate mutated) EMS1 of S. equinus VIT_VB2 in E.coli BL21 (DE3) and studying the gene expression in the new host. The current study deals with the extraction of chromosomal DNA from EMS1 (Ultraviolet and ethyl methyl sulphonate mutated) mutant strain of S.equinus VIT_VB2. The streptokinase (SK) gene was amplified from the mutant strain by cloning matured SK gene (1.2 kb) in pGEX-4T-2 vector which comprises a GST tag and tac promoter. The recombinant plasmid with SK gene was routinely analysed and controlled by polymerase chain reaction. The alignment of gene sequence was analysed with reference restriction enzyme BamHI. Out of 30 recombinant isolates of SK (recombinant clone - RC1 to RC30), 10 isolates expressed the production of SK. The maximum zone of hydrolysis of the rSK from RC10 clone on casein plasminogen overlay agarose assay and fibrin plate method. The activity of fused fibrinolytic protein was determined by the hydrolysis of plasmin using chromogenic substrate assay, it was found to be 12432.51± 1.9 IU mLˉ¹. The partial clot lysis of rSK was found to be 100% at 10th min. The SDS PAGE revealed the molecular weight of GST-tagged SK of 72 kDa. The activity of SK showed 2-fold increase from wild to recombinant strain. Hence this strain could be used industrially for large scale SK production and potential contribution in the field of therapeutics. This study is first to report and to manifest the potential production of recombinant streptokinase from a non-pathogenic strain, Streptococcus equinus isolated from bovine milk.

Login to Download PDF

Please login with your Paper ID and password to access the full PDF.

🔑 Login to Download