title: Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae.
abstract: Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.
authors.affiliation: Transgène S.A., Strasbourg, France.
authors.forename: N
authors.initials: N
authors.lastname: Riehl-Bellon
authors.forename: D
authors.initials: D
authors.lastname: Carvallo
authors.forename: M
authors.initials: M
authors.lastname: Acker
authors.forename: A
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: M
authors.initials: M
authors.lastname: Marquet
authors.forename: G
authors.initials: G
authors.lastname: Loison
authors.forename: Y
authors.initials: Y
authors.lastname: Lemoine
authors.forename: S W
authors.initials: SW
authors.lastname: Brown
authors.forename: M
authors.initials: M
authors.lastname: Courtney
authors.forename: C
authors.initials: C
authors.lastname: Roitsch
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006629:Hirudins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D006629:Hirudins; D011994:Recombinant Proteins