Expression and characterization of antimicrobial peptide ABP-CM4 in methylotrophic yeast Pichia pastoris

Abstract: Antibacterial peptides CM4 (ABP-CM4) is a linear cationic peptide that has antimicrobial properties. To explore a new approach of expression of ABP-CM4 in methylotrophic yeast Pichia pastoris, the gene of ABP-CM4 was obtained by recursive PCR (rPCR) and cloned into the vector pPICZaA. The SacI-linearized plasmid pPICZaA-CM4 was transformed into P. pastoris GS115 by electroporation. The expression was induced about 72h with 0.5% methanol at 20°C, supplied with 2% casamino acids to avoid proteolysis, and approximately 40mg ABP-CM4 was secreted into 1L of medium. Recombinant ABP-CM4 was purified through size-exclusion chromatography and 15mg pure active ABP-CM4 was obtained from 1L culture. Tricine–SDS–PAGE indicated that recombinant ABP-CM4 was secreted as a protein of around 3.8kDa. The recombinant ABP-CM4 appears to be successfully expressed, as it displays antibacterial and antifungal activity (antibacterial assay, polyarylamide gel electrophoresis, and antifungal assay) indistinguishable from those of natural protein. [Copyright &y& Elsevier]