C-terminal proteolytic degradation of recombinant desulfato-hirudin and its mutants in the yeast Saccharomyces cerevisiae.
The potent thrombin inhibitor hirudin variant 1, originally isolated from the leech Hirudo medicinalis, was expressed in Saccharomyces cerevisiae under the control of a truncated glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter fragment. Fusion of the yeast acid phosphatase (PHO5) signal sequence to the hirudin gene led to quantitative secretion of recombinant desulfato-hirudin variant 1 (r-hirudin) into the extracellular medium in a growth-dependent manner. In comparison to the genuine molecule, r-hirudin lacks the sulfate group at the Tyr in position 63. Besides the full-length protein of 65 amino acids (hir65), chemical analysis revealed the presence mainly of two derivatives lacking the last amino acid Gln (hir64) or the penultimate Leu (hir63) in addition. When expressing r-hirudin in mutant strains defective in all but one of the three major known carboxypeptidases, it turned out that the vacuolar carboxypeptidase yscY as well as the alpha-factor precursor-processing carboxypeptidase, ysc alpha, participate in the C-terminal degradation of r-hirudin. Direct involvement of yscY and ysc alpha was confirmed by sequential disruption of their structural genes PRC1 and KEX1, respectively. Disruption of PRA1, coding for the yscY-processing proteinase yscA, also abolished yscY-mediated C-terminal r-hirudin degradation, but clearly reduced the overall expression yield. Since ysc alpha is described to be highly specific for basic amino acids which are not present at the C-terminus of r-hirudin, a series of r-hirudin mutants with changes in the C-terminal amino acids were constructed and analysed for ysc alpha-mediated and yscY-mediated degradation. Chromatographic analysis of the expression products confirmed the preference of ysc alpha for basic amino acids, although Tyr, Leu and Gln were also hydrolysed. It could further be concluded that ysc alpha might also be responsible for the C-terminal degradation of recombinant atrial natriuretic factor and epidermal growth factor expressed in yeast.
Heim
J
J
Ciba-Geigy Ltd., Biotechnology Department, Basel, Switzerland.
Takabayashi
K
K
Meyhack
B
B
Märki
W
W
Pohlig
G
G
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D006629:Hirudins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; C060225:desirudin