title: High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris.
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.
authors.affiliation: Instituto Venezolano de Investigaciones Científicas (IVIC), Laboratorio de Trombosis Experimental, Centro de Biofísica y Bioquímica, Caracas 1020A, Venezuela. merlopez@ivic.ve
authors.forename: Mercedes
authors.initials: M
authors.lastname: Lopez
authors.forename: Anne
authors.initials: A
authors.lastname: Koehler
authors.forename: Goetz
authors.initials: G
authors.lastname: Nowak
mesh_terms: D000991:Antithrombins / Q000378:metabolism; D017931:DNA Primers; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D010843:Pichia / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000991:Antithrombins; D017931:DNA Primers; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C493509:dipetarudin