High-level Expression, Purification And Antibacterial Activity Against Multiple Drug Resistance Bacteria Of Antimicrobial Peptide Pt5e

The antimicrobial peptide Pt5, which consists of the C-terminal 55 residues of zebrafish(Danio rerio) phosvitin (Pv), is indispensable for Pv’s antimicrobial activity. Pt5e, one of the mutants of Pt5 (Arg48 replaced by Leu4), presents a stronger bactericidal activity against E. coli and S. aureus. It has a high research and application value because of its strong bactericidal activity as well as other multiple immune functions without destroying human normal cells. Therefore, Pt5e has a great potential to become a novel antibacterial agent, especially with the prevalence of antibiotics abuse and drug resistant bacteria in modern society.E. coli has been used as the host in the present production strategy for recombinant Pt5e (rPt5e). The His-tag fusion protein has been expressed and purified by Ni-NTA affinity chromatography. However, the yield of rPt5e can hardly meet the needs for further research and exploitation by this heterologous expression and purification system. Thus, the development of another expression system with high-level yield is the key to solving this problem.As a novel tag for the expression and purification of recombinant protein, the lastin-like polypeptides (ELP) tag can make the purification of the target protein easier by centrifuging and significantly increase the expression yield. In this study, a designed cationic elastin-like polypeptide (CELP) tag was described to improve the yield of rPt5e. The antibacterial activity of rPt5e was tested with multi-drug resistance (MDR) bacteria.Firstly, the CELP[KV7F-36] gene was oligomerized with 4 CELP[KV7F-9] genes by recursive directional ligation (RDL). Then the CELP[KV7F-36] gene and the Pt5e gene were modified by inserting enterokinase substrate gene which was used to remove the CELP tag after expression. Followed the construction of the expression vector pET28a-CELP[KV7F-36]-ENK-Pt5e, the fusion protein was expressed in E. coli host by auto-inducing system. And then the fusion protein was purified by inverse transition cycling (ITC) and digested by enterokinase. The rPt5e was released from the fusion protein and purified by the same method. The different yield of rPt5e fusion with CELP tag and His tag were compared. In addition, the bactericidal activity of rPt5e against MDR bacteria was tested.The results showed that the fusion protein and the rPt5e could be purified by ITC successfully after fusion with CELP tag and expression. The yield of rPt5e produced by CELP-tag method was 1.47 mg (from 100 mL culture), which was almost 20 times of that by His-tag method. The increase was significant. Furthermore, rPt5e showed strong antimicrobial activity against the 5 MDR bacteria at a low concentration. It is also observed that rPt5e could destroy the cell wall of the 5 MDR bacteria and kill them by the transmission electron microscope.