We explored the production in Pichia pastoris of a membrane-acting immunotoxin (IT) based on the Cyt2Aa1 toxin from the bacterium Bacillus thuringiensis subspecies kyushuensis. Initial attempts at the P. pastoris expression of Cyt2Aa1 were not successful due to the high A + T-content of the native bacterial gene, resulting in premature transcription termination. Accordingly, we designed and constructed a synthetic cyt2Aa` gene (syncyt2Aa1)(2) that was optimised for expression in this eukaryotic host. This was achieved through a recursive PCR strategy where the overall G + C-content of the cyt2Aa1 DNA sequence was systematically increased to similar to50% compared to similar to30% in the native bacterial gene and only the P. pastoris preferred codons were used. A synthetic DNA sequence coding for a soluble and flexible serine/glycine linker was then used to genetically fuse syncyt2Aa1 with the human single-chain antibody fragment (scFv) C6.5 targeting p185 (HER-2), a cell-surface glycoprotein overexpressed in 30% of human breast and ovarian cancers. Subsequent expression of the resulting IT construct [scFvC6.5-syncyt2Aa1(mychis(6))](2) led to high-level accumulation of the recombinant protein in yeast membranes. Although the solubilisation of scFvC6.5-syncyt2Aa1(mychis(6)) from P. pastoris membranes necessitated the use of guanidine hydrochloride, the use of subsequent in vitro refolding and immobilised metal affinity chromatography (IMAC) steps allowed purification of the recombinant product at yields as high as similar to 10mgl(-1) culture. Despite being core N-linked glycosylated and retaining part of the yeast secretion signal, the P. pastoris produced scFvC6.5-syncyt2Aa1(mychis(6)) exhibited significant specific activity for p185 HER-2 -overexpressing SK-BR-3 cells but not p185(HER-2)-negative Swiss 3T3 cells or human erythrocytes. (C) 2003 Elsevier Science (USA). All rights reserved.