| Project/Area Number |
12490018
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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 |
広領域
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
HAYASHI Takahisa Wood Research Institute, KYOTO UNIVERSITY, Associate professor, 木質科学研究所, 助教授 (70231529)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIYAMA Junji Wood Research Institute, KYOTO UNIVERSITY, Associate professor, 木質科学研究所, 助教授 (40183842)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥11,300,000 (Direct Cost: ¥11,300,000)
Fiscal Year 2001: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2000: ¥7,300,000 (Direct Cost: ¥7,300,000)
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| Keywords | cellulose biosynthesis / GhCesA2 / AxCesA1 / Pichia / insect cells / crystalization / セルロース合成酵素 / アグロバクテリウム / バキュロウィルス |
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| Research Abstract |
GhCesA2 is a cotton (Gossypium hirsutum) homolog of bacterial cellulose synthase gene that encodes cellulose 4-β-glucosyltransferase. The central catalytic region of GhCesA2 was expressed as a soluble protein in methylotrophic yeast Pichia pastoris. The molecular size of the recombinant protein was 100 kDa, which decreased to 85 kDa after the treatment with endoglycosidase H. The recombinant GhCesA2 catalyzed transfer of glucose from UDP-glucose into unknown products in the presence of the extract of cotton hypocotyls, but the products were not β-1,4-glucan. The putative cotton (Gossypium Hirsutum L.) cellulose synthase gene GhCesA2was expressed in insect cells using baculovirus systems and the hydrodynamic properties of the gene product were determined in its native state. The recombinant protein could be solubilized with 1% Triton X-100 from the membrane fraction of infected Sf9 cells. The results obtained from gel filtration chromatography and H_2O/D_2O sucrose gradient sedimentatio
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n showed that the recombinant GhCesA2 had two types in size. We calculated the partial specific volume, Stokes radius and molecular weight of the protein-detergent complex and estimated the molecular weight of the two proteins as 142,000 and 285,000. The result suggested that the recombinant GhCesA2 form a homo-dimer.
Three-dimensional structure of cellulose synthase remains difficult because none of purification and crystallization procedures for its recombinant protein has not been established. In this study, we succeeded in expressing a Histidine-tagged BcsA.(subunit A of a bacterial cellulose synthase) using a bacurovirus expression system in SF9 cells. The expression of this recombinant protein was assessed and verified by SDS-PAGE and western blotting, and found promissing for mass production by purifying them with His tags. We are now stepping in the next stage to measure more precisely the proteins, for instance, the activity as well as the size distribution either by electron microscopy and X-ray small angle scattering. Less
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