Cloning of staphylokinase gene produced from mutant S.aureus in E.coli

This study was aimed to cloning staphylokinase gene from locally isolated Staphylococcus aureus in E.coli and detection the gene expression in new host . Amplified Sak gene was then sequenced to determine the nucleotide sequence of the gene . Results of sequencing showed that there are three point mutation were occurred in the structural gene of staphylokinase according to base pair alignment with the same gene of S.aureus standard strain recorded in BLAST / NCBI . These mutations are miscense mutations doesn’t affect the group of amino acid that was changed then doesn’t affect enzyme tertiary structure and activity . Results of sequencing also showed that the complete nucleotide sequence of the gene doesn’t have a restriction site for BamHI and XbaI that were used for gene cloning . Staphylokinase gene was then double digested with BamHI and XbaI to obtain restriction fragment with sticky ends came from the restriction sites for these two enzymes located in the specific primers designed for this purpose (forward primer containing restriction site for BamHI and reverse primer containing restriction site for XbaI). Then the restriction fragment of staphylokinase gene was ligated in pSP72 cloning vector previously double digested with BamHI and XbaI to get sticky ends compatible with the two sticky ends of Sak gene , then recombinant vector size was 2900 bp.