Purification, Characterization And Gene Cloning Of A Novel Protease With Fibrinolytic Activity From Streptomyces Sp.

A novel fibrinolytic protease from Streptomyces sp.C3662 was purified by (NH₄)₂SO₄ fractionation, DEAE-Sepharose , CM-Sepharose chromatography. The purity of the purified enzyme determined by HPLC was 93.25%. The molecular weight of the protease was indicated to be 25kD by SDS-PAGE. The fibrinolytic activity of the protease is stable in the range of 4-37℃ and pH4.0-9.0, and the optimum is pH7.5. The fibrinolytic activity of the protease was inhibited by 10mmol/L PMFS but wasn't effected by EDTA, which indicated that the protease might be belongs to serine protease class. The activity of the protease on the plasminogen-free fibrin plate indicated that the enzyme is a fibrinolytic enzyme which degrades fibrin directly, and not a plasminogen activator. The blood platelet aggregation test showed that the protease posses antiplatelet aggregation in vitro. The acute toxicity in mouse of the fibrinolytic enzyme was rather low.According to the N-terminal sequence of this protease and the usage preference of synonymous codon in Streptomyces genome, a series of synonymous oligonucleotide probes with the sequence of5'-GTC(G)TGCGGCGGCACG(C)CGC(G)GCC(G)GCC(G)CAG(C)GGC-3' was designed and used to hybridize with total DNA of Streptomyces Sp C3662. A 6.0kb BglII fragment showed positive result and a recombinant plasmid of pGY1 was obtained by cloning into BamHI site in plasmid pUC18. Subcloning and sequencing suggested an ORF consisted of 855bp was present in fragment in the plasmid pGY1. The deduced product of this ORF has high similarity with the glyceraldehyde-3-phosphate dehydrogenase. The failure of obtaining fibrinolytic...