DESIGNING AND EXPRESSION OF THE ENGINEERED SERRALYSIN ENZYME

Background and Aim:Serralysin is a bacterial metalloprotease which is associated with virulence in Serratia mercessence, strain E15. Serralysin is useful in the treatment of pain and inflammation. Serralysin appears to have synergistic effect with antibiotic drugs and augment their antibacterial properties. The serralysin enzyme comprised of 470 amino acids in length with no disulfide bond in the structure. The large structure and possible degradation in the intestines (due to lack of tightened intra-molecular bond) has been cited to possible reduce the bioavailability of the enzyme and large protein structures may not be absorbed well. Therefore, this study is planned in order to design and express the engineered form of serralysin to amend these limitations.Methods:The engineered form of the enzyme with one disulfide bond was designed based on bioinformatics studies and suitable primers were synthesized. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The expression of recombinant protein was confirmed by SDS-PAGE and western blot analysis. Finally the target protein was purified using affinity chromatography method.Results:Amplifying, cloning and expression of the engineered enzyme were accomplished successfully. The protein band was detected on the expected size (approximately 40 KDa) and confirmed with western blot analysis. Purification process leads obtaining properly purified protein.Conclusion:The engineered serralysin was expressed and purified as a recombinant protein in the E. coli properly. However, further analysis should be conducted to evaluate its activity and determining other characteristics.

Keywords:

Engineered serralysin , Cloning , Expression , and Purification