Isolation of recombinant hirudin by preparative high-performance liquid chromatography
Research article published in J Chromatogr (1989)
Abstract
The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described. rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
Three-step HPLC purification of recombinant hirudin variant 2-Lys47 from yeast culture broth; pure product fully active in human alpha-thrombin inhibition assay.
Warum dies für die Hirudotherapie relevant ist
This article describes the purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, using sequential preparative reversed-phase and anion-exchange HPLC steps. The final product was confirmed pure by analytical HPLC, isoelectric focusing, amino acid composition analysis, sequence analysis, and mass spectrometry, and demonstrated full activity in a human alpha-thrombin inhibition assay. Hirudin is referenced directly in the abstract as the target protein, making this relevant to the production and quality control of this anticoagulant protein. CAVEAT: This is a bioprocess engineering and protein chemistry study involving recombinant protein produced in yeast rather than leech-derived material; it includes no leeches, no hirudotherapy application, and no in-vivo data.
Zitation
Isolation of recombinant hirudin by preparative high-performance liquid chromatography.
Bischoff R et al. · J Chromatogr, 1989
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