Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins.
One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.
Idiris
Alimjan
A
ASPEX Division, Research Centre, Asahi Glass Co. Ltd, 1150 Hazawa-cho, Kanagawa-ku, Yokohama 221-8755, Japan.
Bi
Kewei
K
Tohda
Hideki
H
Kumagai
Hiromichi
H
Giga-Hama
Yuko
Y
D001709:Biotechnology / Q000379:methods; D015139:Blotting, Southern; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010447:Peptide Hydrolases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D019382:Human Growth Hormone; D010447:Peptide Hydrolases