Expression of recombinant streptokinase (SKA) of group A streptococci in E. coli

authors:

avatar N Molaee , avatar H Abtahi 1 , * , avatar Gh Mossayebi

,Arak University of Medical Sciences, Arak, Iran
Journal of Inflammatory Diseases: Vol.16, issue 3; 11-18
article type: issue_documents_importer

how to cite: Molaee N, Abtahi H, Mossayebi G. Expression of recombinant streptokinase (SKA) of group A streptococci in E. coli. J Inflamm Dis. 2012;16(3):e155704. 

Abstract

    Background: Streptokinase A is an antigenic protein secreted by Streptococcus pyogenes. This protein can be used to liquefy pus in pneumonia and the purulent joint swelling and also as an antigen for detection of group A streptococcal infections.   Objective: The purpose of this study was expression and production of recombinant streptokinase A of group A streptococci in Escherichia coli in line with diagnostic and therapeutic purposes.   Methods: Streptokinase A gene was initially amplified by polymerase chain reaction (PCR) method and sub-cloned into prokaryotic expression vector pET32a. Later, the pET32a vector was transformed into Escherichia coli BL21-DE3-plySs. Gene expression product, induced by IPTG, was purified by Ni-NTA purification kit, and measured by Bradford method. Recombinant SKA was further analyzed by Western Blot. Gene was amplified and sequenced using the Sanger method and the amplified gene in plasmid pTZ57R / T were identical to Recorded sequence in gene bank for streptokinase gene A .   Findings: The nucleotide sequence of the gene amplified by PCR was determined by Sanger method. Sequencing results showed similarity in nucleotide sequence of the cloned gene in E. coli with that of group A streptococci available in GeneBank database. The amount of protein product obtained by Bradford method was 3 mg/ml. Recombinant streptokinase protein reacted with mouse sera containing anti-streptokinase A.   Conclusion: Our data showed that expression of recombinant SKA protein is possible in Escherichia coli host. The protein product had an approximate molecular weight of 67 kDa with its antigenic properties unchanged. Thus, it can be substituted for ASO and SLO tests used in diagnosis of patients with group A streptococcal infections.