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
This study reports the development of a recombinant Saccharomyces cerevisiae strain expressing a hybrid HV1-HV2 hirudin gene, achieving secretion of 44 mg/L of recombinant hirudin after galactose induction, with a total purification yield of 3.6 g at >96% polypeptide purity. The hybrid rHIR HV1-HV2 protein exhibited essentially equipotent anticoagulant activity and thrombin-binding affinity compared to previously reported HV1 and HV2 preparations, as measured by activated partial thromboplastin time and thrombin:hirudin inhibition reaction. This is directly relevant to ASH's domain, addressing large-scale production and characterization of the primary bioactive anticoagulant molecule from medicinal leech saliva for potential therapeutic use. However, the study is a bioprocess development and biochemical characterization effort with no animal or clinical data, and no leeches are directly involved.
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: June 18, 2026