Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae
Research article published in Protein expression and purification (1993)
Abstract
Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents. A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin. Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction. rHIR was purified by a process that consisted of two chromatographic steps and decolorization. Total yield for the purification process was 3.6 g, or 41%. This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography. Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA. Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents.
Por qué esto importa para la hirudoterapia
Este estudio describe el desarrollo de una cepa recombinante de Saccharomyces cerevisiae que expresa un gen híbrido de hirudina HV1-HV2, logrando la secreción de 44 mg/L de hirudina recombinante tras la inducción con galactosa, con un rendimiento total de purificación de 3,6 g con una pureza polipeptídica >96%. La proteína híbrida rHIR HV1-HV2 mostró una actividad anticoagulante y una afinidad de unión a la trombina esencialmente equipotentes en comparación con preparaciones de HV1 y HV2 previamente descritas, según las mediciones mediante el tiempo de tromboplastina parcial activada y la reacción de inhibición trombina:hirudina. Esto es directamente relevante para el ámbito de la ASH, ya que aborda la producción a gran escala y la caracterización de la principal molécula bioactiva anticoagulante de la saliva de la sanguijuela medicinal para un posible uso terapéutico. No obstante, el estudio es un trabajo de desarrollo de bioprocesos y caracterización bioquímica sin datos animales ni clínicos, y no intervienen directamente sanguijuelas.
Citación
Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae
Lehman ED et al. · Protein expression and purification, 1993
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026