| Project/Area Number |
09460156
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SAKAI Yasuyoshi Kyoto University, Graduate School of Agriculture, Associate Professor, 農学研究科, 助教授 (60202082)
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| Co-Investigator(Kenkyū-buntansha) |
MURATA Masayuki National Institute for Physiological Sciences, Department of Molecular Physiology, Associate Professor, 生理学研究所, 助教授 (50212254)
YURIMOTO Hiroya Kyoto University, Graduate School of Agriculture, Assistant Professor, 農学研究科, 助手 (00283648)
KATO Nobuo Kyoto University, Graduate School of Agriculture, Professor, 農学研究科, 教授 (50026556)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | Methylotrophic yeasts / Biotraffic engineering / Candida boidinii / Peroxisomes / Heterologous gene expression / Protein traffic / Autophagy / Oxidases / 細胞内タンパク質輸送工学 / オルガネラ / Pichia pastoris / 液胞 / タンパクの選別輸送 / カタラーゼ / ソーティングシグナル |
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| Research Abstract |
The methylotrophic yeast is widely used as a heterologous gene expression system in academic and applied fields. When cells are grown on methanol, a large volume of cells (up to 80%) was occupied by a single-membrane bound organelle, the peroxisome. The study was conducted to produce toxic oxidases within peroxisomes of the methylotrophic yeast, since many oxidases are useful for clinical diagnosis. To this end, the protein traffic was manipulated for efficient protein transport, and this strategy (or concept) was named "sorting engineering" (later biotraffic engineering). Since genetic diorders deficient in peroxisome assembly are known, the molecular mechanism for protein traffic to peroxisomes was in important in medical field and investigated using the methylotrophic yeast as a model organism. In this study, the information obtained from the basic research was applied to production of fungal fructosyl amino acid oxidase, which is useful for determination of glycated proteins. In diabetic patients, the level of glycated proteins is high. Using (i) an efficient targeting signal to peroxisomes and (ii) an alcohol-oxidase depleted strain, and (iii) optimizing codon usage and culture conditions, we could improve the enzyme productivity approximately 47-fold from the orginal transformant. The produced protein accumulated within membrane-bound peroxisomes up to nearly 20% of the toal soluble protein. This is the first successful example which was conducted based on the concept "sorting engineering" ("biotraffic engineering"), and a similar approach will be possible in any other eukaryotic cells for heterologous gene expression, especially for the production of toxic or membrane proteins.
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