| Project/Area Number |
02454033
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | Osaka University |
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Principal Investigator |
HARASHIMA Satoshi Osaka University, Department of Biotechnology Associate Professor, 工学部, 助教授 (70116086)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Heterol gous expression / Yeast promoter / Host mutation / Transcription / nucleosome / chromatin / Brewing yeast / Temperature-regulatable expression system / 転写調節 / 突然変異株 / 発現調節 |
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| Research Abstract |
Toward to construction of Saccharomyces cerevisiae super host for efficient expression of heterologous proteins, mutants designated bel (basal expression level) showing increased production of reporter protein. Analysis of bel mutants revealed that bel mutations caused increased transcription of various genes through affecting chromatin or higher order of chromosomal organization. It was suggested that the use of bel mutation could lead to further increased promoter activity of some set of genes.
From the screening of mutants displaying disregulation in mating-type control in S. cerevisiae, a novel mutations designated hml alpha 2-102 was isolated. In combination of hml alpha 2-102 mutation with a temperature sensitive mutation in the SIR3 gene which acts to repress the expression of silent copy of mating-type informations residing at HML and HMR locus, a system for temperature-controlled expression of a foreign gene with dual mode was developed.
The PH084 gene was cloned and found to encode an inorganic phosphate (Pi) transporter in S. cerevisiae. Analysis of transcriptin of PH084 revealed that PHO84 is strongly derepressed under Pi starved condition. Combining of promoter of PHO84 with a temperature sensitive mutation in PHO81 which is necessary for expression of the PHO84 gene led to the establishment of another temperature regulatable expression system. Using this system, human lysozyme and rice alpha -amylase was efficiently produced.
Aiming at the use of brewing strains as host for production of heterologous proteins, transformation system of prototrophic brewing strains using a dominant selective marker in combination with cerulenin was developed.
All of these studies provided useful informations for breeding of super host cell of S. cerevisiae for efficient production of heterologous proteins.
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