Optimized Expression and Characterization of Antimicrobial Peptides, LPcin Analogs

Abstract

Lactophoricin (LPcin or LPcin‐I), a cationic amphipathic antimicrobial peptide consisting of 23 amino acids, is a part of proteose peptone (113–135) isolated from bovine milk. The 23 amino acids of LPcin are relatively long for industrial uses as antimicrobial peptides. Therefore, a series of N‐ or C‐terminal truncated variant mutated analogs and truncated mutated analogs were designed using peptide engineering techniques to investigate the structure–activity relationships. Then, three analogs of LPcin‐C8 [(LPcin‐YK1), LPcin‐T2WT6W (LPcin‐YK2), and LPcin‐T2WT6W‐C8 (LPcin‐YK3)] were optimized to express with high yield and characterized for their structural studies. Successful expression of these three LPcin analogs in Escherichia coli system was accomplished through the formation of a fusion protein with higher yield. The peptides were purified using several biophysical techniques and finally isolated using reversed‐phase preparative HPLC. The purity, molecular mass, and the secondary structure of the purified and isolated LPcin analogs were characterized by polyacrylamide gel electrophoresis, mass spectrometry, and circular dichroism spectrometry. The secondary structure of LPcin and its three analog peptides clearly showed α‐helical characteristics in membrane environments and random‐coil conformation in water. Consequently, our results so far have shown that the three LPcin analogs could become potent antimicrobial peptides like LPcin as an alternative of new antibiotics in the near future.