A small multifunctional cytokine, growth-blocking peptide (GBP), front the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem. we utilized a high-density fermentation method. The pH Of the medium in the fermenter was kept at 3.0, and the medium was collected within 48 h post methanol shift to minimize exposure of the target peptide to proteases, Recombinant GBP was purified through three reverse-phase HPLC columns, We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar, between chemically synthesized GBP and purified recombinant GBP. Up to 50 mg GBP was recovered pet, 1 L of yeast culture supernatant. (C) 2002 Elsevier Science (USA). All rights reserved.