CLONING AND EXPRESSION OF TRUCATED FORM OF SERRALYSIN ENZYME IN ESCHERICHIA COLI

Background and Aim:Serralysin is a proteolytic enzyme with anti-inflammatory, fibrinolytic and anti-biofilm activity. Nowadays due to spread of the antibiotic resistance, the roll of serralysin for destroying biofilms is highlighted. The big length of the protein which hampers its penetration is the main drawback of it. It was suggested that the main strategy for increasing its biological efficiency is altering the structure without destroying the catalytic activity. Therefore, this study is planned in order to design and express the truncated form of serralysin enzyme in E.coliMethods:Based on the bioinformatics studies the truncated form was designed and suitable primers were synthesized. The cloning procedure was done by using NcoI and XhoI restriction enzymes and pET28a expression vector. Recombinant plasmid was transformed into E.coli DH5a as a cloning host and then sub cloned into E.coli BL21 as an expression host. Cloning was confirmed by colony PCR and double restriction enzyme digestion. The recombinant enzyme expression was confirmed by SDS-PAGE and western blot analysis. Then the target protein was purified using Ni-NTA chromatography.Results:: Cloning and expression of the recombinant truncated serralysin was confirmed by detecting the protein band on the expected size. The optimal conditions of expression was detected in the LB broth, at 37C, IPTG 1 mM , OD600: 0.6 and expression for 4 hours.Conclusion:: Based on the results, the designed truncated form of the serralysin was successfully expressed and purified as a recombinant protein in the E. coli. Additional analysis should be accompanied to evaluate its activity and other characteristics.

کلیدواژه ها:

Serralysin ، Truncated form ، Recombinant protein ، Anti biofilm