Construction of a protease‐deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease‐sensitive heterologous proteins

• Published in: Yeast (Chichester, England) Vol. 23; no. 2; pp. 83 - 99
• Main Authors: Idiris, Alimjan, Bi, Kewei, Tohda, Hideki, Kumagai, Hiromichi, Giga‐Hama, Yuko
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 30.01.2006
• Subjects: Biotechnology - methods; Blotting, Southern; disruptant; DNA, Fungal - chemistry; DNA, Fungal - genetics; heterologous expression; human growth hormone; Human Growth Hormone - biosynthesis; Human Growth Hormone - genetics; Human Growth Hormone - metabolism; Mutagenesis, Insertional; Peptide Hydrolases - deficiency; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Polymerase Chain Reaction; protease; proteolytic degradation; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Schizosaccharomyces - enzymology; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/yea.1342

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. Copyright © 2006 John Wiley & Sons, Ltd.