Cloning, expression and characterization of staphylokinase in Escherichia coli

Abstract

Staphylokinase (SAK) is a protein secreted from certain strains of Staphylococcus aureus. Staphylokinase is a plasminogen activator, which converts plasminogen to plasmin which decomposes fibrin clots. Staphylokinase gene has been cloned from chromosomal DNA of Staphylococcus aureus NCTC 10033 by polymerase chain reaction(PCR). The gene was subcloned into pUC19 and confirmed by nucleotide sequencing. The gene consisted of 408 base pairs encoding 136 amino acids. The staphylokinse gene was inserted into pKK-omp vector having a tac promoter and an ompA signal sequence. The resulting expression vector for staphylokinase was named pSAK. Expression of recombinant staphylokinase was qualitatively confirmed by skim-milk human plasminogen overlay test. Escherichia coli JM109 carrying pSAK expressed and secreted about 15mg/liter of recombinant staphylokinase into periplasmic space. The recombinant staphylokinase in the periplasm of E. coli JM109 was purified by using cold osmotic shock, DEAE-chromatography and CM-chromatography. The purified recombinant staphylokinase had the same amino acid sequence in the N-terminal region and plasminogen-activating activity as authentic staphylokinase. The recombinant staphylokinase was modified with methoxypolyethyleneglycol(mPEG) to reduce its antigenicity for therapeutic applications. In enzyme-linked immunosorbent assay (ELISA), mPEG-modified staphylokinse showed lower immuno-reactivity than that of unmodified staphylokinase