Expression of recombinant hybrid peptide cecropinA(1–8)–magainin2(1–12) in Pichia pastoris: Purification and characterization

Hybrid antibacterial peptide CA–MA (cecropinA(1–8)–magainin2(1–12)) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach of expression of hybrid peptide CA–MA in methylotrophic yeast, Pichia pastoris, the gene of CA–MA was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The SalI-linearized plasmid pPICZα-CA–MA was transformed into P. pastoris SMD1168 by electroporation. The expression was induced for 96 h with 1.0% methanol at 28 °C, pH 5.0. Recombinant CA–MA was purified by reversed-phase HPLC and 22 mg pure active CA–MA was obtained from 1 L fermentation culture. Tricine–SDS–PAGE indicated that recombinant CA–MA protein molecular weight is 2.6 kDa. Mass spectrometry of purified CA–MA demonstrated a single large signal for the molecular ion [M+2H+]2+ at 1281.07 m/z, identical to that of the putative protein (2.56 kDa). Antimicrobial assays showed that CA–MA has a broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. This is the first report on the heterologous expression of a hybrid antibacterial peptide with molecular weight below 3.0 kDa in P. pastoris. Our results demonstrate that functional CA–MA can be produced in sufficient quantities using P. pastoris for use in further studies on functionality and diagnostic applications.