期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 77, 期 12, 页码 2461-2466出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.130588
关键词
glucan engineering; protease-deficient strain; mutagenesis; yeast
类别
资金
- New Energy and Industrial Technology Development Organization (NEDO) of Japan
- KAKENHI [23780097]
- Grants-in-Aid for Scientific Research [23780097] Funding Source: KAKEN
Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man(5)GlcNAc(2), an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man(8)GlcNAc(2) to Man(5)GlcNAc(2) was observed after integration of Aspergillus saitoi alpha-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-beta by approximately 10-fold.
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