| Project/Area Number |
05453160
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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 |
応用微生物学・応用生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
TAKAGI Masamichi The University of Tokyo, Department of Biotechnology, Professor, 農学部, 教授 (50018339)
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| Co-Investigator(Kenkyū-buntansha) |
HORIUCHI Hiroyuki The University of Tokyo, Department of Biotechnology, Assistant Professor, 農学部, 助手 (00209280)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1993: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | Filamentous Fungi / Aspartic Proteinase / Chitinase / Prosequence / Rhizopus / Folding / Secretion / aspartic proteinase / molecular chaperone / S.cerevisiae / pro-sequence / secretion / Rhizopus niveus |
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| Research Abstract |
Rhizopus niveus aspartic proteinse-I (RNAP-I) has the prosequence at its N-terminus and could be secreted efficiently from Saccharomyces cerevisiae. Purified RNAP-I with the prosequence once denatured in 6M guanidine HCl could be renatured and activated to have 75% of the enzymatic activity by removing guanidine HCl in vitro, but RNAP-I without the prosequence (mature RNAP-I) could not. The wild-type prosequence helped the recovery of the activity of the denatured mature RNAP-I at the level of 70% in trans. From these results, we concluded that the prosequence of RNAP-I guides correct folding of its mature part. The site of degradation of RNAP-Is with mutated prosequences which were not secreted from S.cerevisiae in the cell was investigated with indirect immunofluorescent microscopy, with sec mutants, and by cell fractionation analysis. It is elucidated that RNAP-Is with mutated prosequences were degraded in the endoplasmic reticulum.
Chitinase 1 of R.oligosporus has prosequence at its C-terminus and is secreted extracellularly. Chitinase 1 without the prosequence (mature chil) could be secreted efficiently from S.cerevisiae. Chitinase l with the prosequence (prochil) has very weak enzymatic activity. From the results of pulse chase experiment and cell factionation analysis, it is suggested that prochil is transported to the cell wall after its synthesis and that converted to the mature chil when the cells autolyse.
To investigate the function of the prosequence of RNAP-I and chitinase 1 in Rhizopus, we have also established the transformation system of R delemar and succeeded in the expression of heterologous gene in R delemar.
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