Cloning and overexpression of active recombinant fusion streptokinase: a new approach to facilitate purification.
Streptokinase is a common fibrinolytic drug and included in the World Health Organization (WHO) Model List of Essential Medicines. Comparative clinical trails such as cost-effectiveness suggest that streptokinase can be the drug of choice for thrombolytic therapy. To reach the highest amount of the protein and production of active form of streptokinase in bacteria need to modify and optimize methods. In the present study, chromosomal DNA was extracted from S. equisimilis H46A and used for amplification of streptokinase gene (skc) (mature section: 1245 bp) by cloning into pGEX-4T-2 vector which contains a tac promoter. The cloning results were controlled by PCR, double digestion and sequencing. The expression level of the protein in different strain of E. coli was optimized and reached up to 50% of the total cell protein. The function of the fusion protein as active fibrinolytic protein was confirmed by plasmin hydrolysis of chromogenic peptidyl anilide substrate assay.
Nejadmoghaddam
Mohammad Reza
MR
Department of Recombinant Technology, Nanobiothecnology Research Center, Avesina Research Institute, Tehran, Iran.
Modarressi
Mohammad Hossein
MH
Babashamsi
Mohammad
M
Chamankhah
Mahmood
M
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D013291:Streptococcus / Q000235:genetics; D013300:Streptokinase / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D015912:Thrombolytic Therapy
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013300:Streptokinase