Cytoplasmic Expression, Optimization and Catalytic Activity Evaluation of Recombinant Mature Lysostaphin as an Anti-staphylococcal Therapeutic in Escherichia coli
Objective: Scientists are to develop novel antibacterial therapeutics in order to eliminate medically significant pathogens resistant to antibiotic. Lysostaphina zinc metalloprotease with the sub-atomic weight of 27kDa and particularly tic action against Staphylococcus aureus degrades the S. aureus by hydrolyzing the pentaglycine cross-links introduce in its cell wall. Because of such potential, lysostaphin will be a decent agent for treatment of antibiotic-resistant staphylococcal infections. Also, due to the broad needs of society to increase localization of sciences, the national production of this drug in research laboratories would be necessary. Materials and Methods: First, with the aid of articles we identified Lysostaphin sequences without signal peptide via Gene Bank data base. In order to achieve the correct protein sequence, we add the entrokinase cutting site to our sequence. To ensure the correct cloning process, the entire process of cloning and protein expression was evaluated in silico. The sequence obtained for the synthesis was sent to the Biomatik Company in Canada (BIOMATIK). After the primer design and synthesis of the DNA fragment of the gene, we amplified the gene using PCR. The mature lysostaphin gene was cloned in plasmidpET28a and expressed in E.coli with the carboxyl terminal hexa‑histidine fusion tag under the transcriptional control of T7/lac promoter/operator. Result: The transformed E.coli BL2 (DE3) cells produced catalytically active recombinant lyso staphin after being induced by IPTG. Conclusion: This study shows that the E. coli expression system is suitable for expression of recombinantly so staphin and according to the conducted bioassay in this study, the expressed protein can be considered as an effective therapeutic agent against antibiotic resistant staphylococcus aureus.