Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae.
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.
Riehl-Bellon
N
N
Transgène S.A., Strasbourg, France.
Carvallo
D
D
Acker
M
M
Van Dorsselaer
A
A
Marquet
M
M
Loison
G
G
Lemoine
Y
Y
Brown
S W
SW
Courtney
M
M
Roitsch
C
C
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*
D006629:Hirudins; D011994:Recombinant Proteins