Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin 11 and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His(+) transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8 h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 11 culture supernatant. This yield is 500-fold higher than the yield obtained with the E coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The K-i value of dipetarudin was 399 83 M, which is in agreement with that calculated for the inhibitor isolated from E coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations. (c) 2006 Elsevier Inc. All rights reserved.