Preliminary Study On Molecular Cloning, Expression And Antibacterial Activity Of Antimicrobial Peptide NK-2

Antimicrobial peptides (AMPs) are a class of small peptides secreted by immune system with antibacterial, antiviral and/or antitumor activities. Although natural AMPs can be isolated from biological materials, both complicated purification procedure and low concentration limit their application in clinic. AMPs can also be synthesized chemically, but their production costs are very high. With the quick development of recombinant DNA technology, AMPs are now can be produced at low costs.The antibacterial peptide NK-2 is a positively charged core segment of NK-lysin with a molecular mass of 3.2kDa and a broad spectrum of antibacterial activities. To develop an efficient expression system for production of recombinant NK-2 at low cost, Sce and Ssp intein sequences were amplified from prokaryotic expression vectors pTYB11 and pTWIN1 and subcloned into expression vector pET-EIGFP after removal of△I-CM intein and GFP sequences by restriction digestion, resulting in two new vectors called pET-ESI1 and pET-ESI2, respectively. NK-2 sequence was amplified by overlapping PCR and subcloned into pET-ESI2 vector. After induction at low temperature, a fusion protein of expected size was detected in the recombinant E.coli using SDS-PAGE. By using the temperature-induced reversible inverse transition property of ELP, the fusion protein was purified by repeated centrifugation at different temperatures in the presence of 0.4M (NH4)2SO4. By using the autocleavage property of intein, the recombinant NK-2 was partially released from the fusion tag with antibacterial activity against DH5αE.coli and Staphylococcus areus.