Expression and identification of an antimicrobial peptide VIP in Pichia pastoris

Abstract:

With the sequence of the vasoactive intestinal peptiepeptide (VIP) from humans and according to the condon bias of Pichia pastoris, we designed PCR primers of VIP and obtained the sequence of VIP by SOE-PCR. Then VIP gene was cloned into Pichia pastoris secretory expression vector and the cell secretary system GS115-pPICZαA-vip was constructed. The recombinant strain was induced by methanol for 96 hours, and we collected the supernatant and identified the VIP by mass spectrometry. The molecular weight of VIP was consistent with theoretical molecular weight. The final result showed that the target peptide VIP was successfully expressed. The experimental investigations of agarose gel diffusion revealed that the recombinant expression modified VIP had relatively strong antibacterial activity to E. coli ATCC25922 and S. aureus ATCC25923. The minimal inhibitory concentration (MIC) of VIP to E. coli ATCC25922 and S. aureus ATCC25923 was 8 mmol/L and 16 mmol/L. Further cytotoxicity and hemolytic experiments indicated that recombinant VIP was non-toxic to normal cells NCM460 and IPEC-J2, had little hemolysis activity to SD rat erythrocytes. Meanwhile, by transmission electron microscopy, we found that VIP mainly inhibited bacteria by disrupting the cell membrane. These experiments established a useful system for further studies, application and mass production of antimicrobial peptide VIP.