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 Sacl-linearized plasmid pPICZaA-CM4 was transformed into R pastoris GS 115 by electroporation. The expression was induced about 72 h with 0.5% methanol at 20 degrees C, supplied with 2% casamino acids to avoid proteolysis, and approximately 40 mg ABP-CM4 was secreted into 1L of medium. Recombinant ABP-CM4 was purified through size-exclusion chromatography and 15 mg pure active ABP-CM4 was obtained from 1L culture. Tricine-SDS-PAGE indicated that recombinant ABP-CM4 was secreted as a protein of around 3.8 kDa. 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. (c) 2005 Elsevier Ltd. All rights reserved.