High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris
Research article published in Protein Expr Purif (2006)
Abstract
Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II 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 8h 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 1l 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 Ki value of dipetarudin was 399+/-83 fM, 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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
Dipetarudin, a chimera between dipetalogastin II and hirudin, expressed in Pichia pastoris at 150 mg/L purified yield (500-fold higher than E.
Why This Matters for Hirudotherapy
This study reports high-level secretion of dipetarudin—a genetically engineered chimeric direct thrombin inhibitor combining dipetalogastin II and hirudin—in Pichia pastoris, achieving approximately 150 mg purified protein per liter of culture supernatant, a 500-fold improvement over E. coli. The purified product had a MALDI-TOF mass of 7450 Da consistent with correct signal sequence processing and a Ki of 399 ± 83 fM for thrombin inhibition, matching the E. coli-derived inhibitor. For ASH, this is relevant as dipetarudin incorporates hirudin as a chimera component and the study addresses cost-effective large-scale production. Caveat: This is a bioprocess/expression-system optimization study; it contains no animal, clinical, or in vivo pharmacological data, and relevance is to recombinant production technology rather than hirudotherapy.
Citation
High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris.
Lopez M et al. · Protein Expr Purif, 2006
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