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.
Resumen
Dipetarudin, a chimera between dipetalogastin II and hirudin, expressed in Pichia pastoris at 150 mg/L purified yield (500-fold higher than E.
Por qué esto importa para la hirudoterapia
Este estudio describe la secreción a alto nivel de dipetarudina —un inhibidor directo de la trombina quimérico obtenido por ingeniería genética que combina dipetalogastina II e hirudina— en Pichia pastoris, alcanzado aproximadamente 150 mg de proteína purificada por litro de sobrenadante de cultivo, una mejora de 500 veces respecto a E. coli. El producto purificado presentó una masa por MALDI-TOF de 7450 Da, coherente con un procesamiento correcto de la secuencia señal, y un Ki de 399 ± 83 fM para la inhibición de la trombina, equiparable al del inhibidor derivado de E. coli. Para ASH, esto es relevante dado que la dipetarudina incorpora hirudina como componente quimérico y el estudio aborda la producción a gran escala de forma rentable. Salvedad: se trata de un estudio de optimización de bioproceso/sistema de expresión; no contiene datos farmacológicos en animales, clínicos ni in vivo, y su relevancia se refiere a la tecnología de producción recombinante más que a la hirudoterapia.
Citación
High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris.
Lopez M et al. · Protein Expr Purif, 2006
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026