Streptokinase (SK) (EC 3.4.99.22) is an extracellular protein produced by several strains of beta-hemolytic streptococci and an activator for conversion of plasminogen to plasmin that hydrolyzes fibrin of thrombi. SK is used to treat blood vessel blockage by blood clots. SK (47 kDa) is a single polypeptide containing 414 amino acids. In a previous study, the mature gene msk from Streptococcus pyogenes (1245 bp, GenBank: EU872448) was cloned and overexpressed in E. coli BL21 (DE3) with 6xhis tidine tag which might cause some side effects to human. In this study, the authors expressed the SK without 6xhis tag, similar to the wild type form. Primers for the gene SK from S. pyogenes were designed with two restriction enzymes Ndel and Xhol at both ends of primers and the stop codon was upstream the 6xhis tag. The gene SK was cloned into pJET1.2 blunt resulting in the pJmSK, cut out from pJmSK with Ndel and Xhol, and inserted into pET21a+ also within Ndel and Xhol resulting in the recombinant plasmid pEmSK, which was transformed into E. coli JM109 (DE3) and expressed at a level 3320 U/ml culture broth.