The Staphylokinase Secretion Expression And Preliminary Purification, In The Variable Lead Green Streptomyces

Thrombotic complications of cardiovascular diseases are main cause of death and disability. Thrombolytic agents currently in use, which act directly or indirectly as plasminogen activator , can effectively preserve heart function and improve survival. Compared with those thrombolytic agents using in clinic, Staphylokinase (SAK), a 15KD profibrinolytic protein produced by certain strains of Staphylococcus aureus. has promising properties including: high thrombolytic efficiency especially for the platelet-rich thrombi; rapid speed of thrombolytic action; remarkable fibrin-specificity and presumed low cost of production by recombinant DNA technology.In this paper, With total DNA extracted from S. aureus in clinic as template, the 730bp DNA fragment including structure gene of SAK, SD sequence and -35-10 sequence of SAK was amplified by PCR. The PCR product was then cloned into E.coli plasmid pUC18 and sequenced This 730bp DNA fragment was proved by sequence analysis to be exactly the same as that of the Sak42D gene reported before. The SAK gene was inserted into Streptomyces lividans plasmid pIJ487 polycloning site to construct recombinant plasmid pIJ487SAK, in which the transcription of the SAK gene was directed by its own promoters. Another approach was made to constructed recombinant plasmid pIJ459SAK by inserting the SAK gene into plasmid pIJ459, which has erm promoter upstream of the pIJ487 polycloning site. So the transcription of SAK gene in pIJ459SAK was direct by both the erm promoter and the promoter in SAK geneTwo recombinant strains harboring recombinant plasmid pIJ487SAK and pIJ459SAK were obtained by transforming the two recombinant plasmids into S.lividans TK54 respectively. The thrombolytic activity of the...