Functional roles of prosequences in the ability of filamentous fungi to secrete a large amount of proteins

原序列在丝状真菌分泌大量蛋白质能力中的功能作用

基本信息

  • 批准号:
    05453160
  • 负责人:
  • 金额:
    $ 4.8万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for General Scientific Research (B)
  • 财政年份:
    1993
  • 资助国家:
    日本
  • 起止时间:
    1993 至 1994
  • 项目状态:
    已结题

项目摘要

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.
牛根霉天冬氨酸蛋白- i (RNAP-I)在其n端有序列,可从酿酒酵母中高效分泌。纯化的rnap - 1在6M胍HCl中变性后,在体外脱除胍HCl后,其酶活性可达到75%,而未脱除前序的rnap - 1(成熟的rnap - 1)则不能。野生型序列帮助变性成熟rnap - 1的活性恢复到70%的水平。从这些结果我们得出结论,rnap - 1的序列引导其成熟部分的正确折叠。用间接免疫荧光显微镜、多突变体和细胞分离分析研究了非酿酒酵母分泌的突变序列的RNAP-Is在细胞中的降解位点。结果表明,具有突变序列的RNAP-Is在内质网中被降解。几丁质酶1在其c端有序列,并在细胞外分泌。几丁质酶1无基因序列(成熟子代)可在酿酒酵母中高效分泌。几丁质酶1具有很弱的酶活性。从脉冲追踪实验和细胞分裂分析的结果来看,丙二醇在合成后被转运到细胞壁,并在细胞自溶时转化为成熟产物。为了研究rnap - 1序列和几丁质酶1在根霉中的功能,我们还建立了R delemar的转化体系,并成功地在R delemar中表达了外源基因。

项目成果

期刊论文数量(24)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Masamichi Takagi and Hiroyuki Horiuchi: "Rhizopus niveus.In : Food Biotechnology : Microorganisms." VCH Publishers, New York. 535-548 (1995)
Masamichi Takagi 和 Hiroyuki Horiuchi:“Rhizopus niveus.In:食品生物技术:微生物。”
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    0
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堀内裕之 他(分担執筆): "真核微生物のキチナーゼ遺伝子「キチン、キトサンハンドブック」" 技報堂(印刷中), (1995)
Hiroyuki Horiuchi 等人(合著者):“真核微生物的几丁质酶基因:“几丁质和壳聚糖手册””Gihodo(印刷中),(1995)
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    0
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堀内裕之 他: "Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar." Curr.Genet.(印刷中).
Hiroyuki Horiuchi 等人:“尼维根霉pyr4 基因的克隆及其在德莱马根霉转化中的应用”(正在出版)。
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    0
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福田良一 他(分担執筆): "酵母の小胞体膜と蛋白質分泌「酵母とバイオ-酵母研究の新潮流-」" 医学出版センター, 248-266 (1994)
Ryoichi Fukuda 等人(合著者):“酵母内质网膜和蛋白质分泌‘酵母和酵母研究的生物新趋势’”医学出版中心,248-266(1994)
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    0
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Hiroyuki Horiuchi and Masamichi Takagi: "Shinkakubiseibutsu no chitinase idenshi "Chitin, chitosan handbook"" Gihoudou, Tokyo. (in press) (in Japanese). (1995)
Hiroyuki Horiuchi 和 Masamichi Takagi:“Shinkakubiseibutsu no chitinase idenshi“甲壳素,壳聚糖手册””Gihoudou,东京。
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TAKAGI Masamichi其他文献

TAKAGI Masamichi的其他文献

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{{ truncateString('TAKAGI Masamichi', 18)}}的其他基金

Construction of effective PCB-degradation bioreactor by modification of regulatory system of PCB-degrading bacterium using genetic engineering.
利用基因工程改造PCB降解菌调控系统构建有效的PCB降解生物反应器。
  • 批准号:
    09556015
  • 财政年份:
    1997
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Studies on structure-function relationship of P450-multi-gene family from yeast and regulatory mechanism of their expression
酵母P450多基因家族结构功能关系及其表达调控机制研究
  • 批准号:
    08406009
  • 财政年份:
    1996
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Breeding of plants resistant to fungal infections by introduction of a chitinase gene from filamentous fungi.
通过引入丝状真菌的几丁质酶基因来培育抗真菌感染的植物。
  • 批准号:
    07556021
  • 财政年份:
    1995
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Tip Growth of Plant-Pathogenic Fungi and Tip-Chitin Metabolism
植物病原真菌的尖端生长和尖端甲壳素代谢
  • 批准号:
    06044062
  • 财政年份:
    1994
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for international Scientific Research
On the Regulation of Membrane Biogenesis by Using Yeast Model Systems
利用酵母模型系统调控膜生物发生
  • 批准号:
    05044123
  • 财政年份:
    1993
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for international Scientific Research
Cycloheximide-resicetance gene isolated from Candida maltosa : Mechanism of action and its utilization
麦芽糖念珠菌中分离的放线菌酮抗性基因:作用机制及其利用
  • 批准号:
    61470129
  • 财政年份:
    1986
  • 资助金额:
    $ 4.8万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (B)

相似海外基金

Structure-Function Relationships of Potato Aspartic Proteinase
马铃薯天冬氨酸蛋白酶的结构-功能关系
  • 批准号:
    416251-2011
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Structure-function relationships of potato aspartic proteinase
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    400716-2010
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    2010
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Characterization of a novel aspartic proteinase and development of its inhibitor
新型天冬氨酸蛋白酶的表征及其抑制剂的开发
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    12557085
  • 财政年份:
    2000
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    $ 4.8万
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Functional analysis of rice aspartic proteinase, oryzasin and application to milk clotting
大米天冬氨酸蛋白酶、米扎星的功能分析及其在凝乳中的应用
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    10680160
  • 财政年份:
    1998
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    $ 4.8万
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PLASMODIUM FALCIPARUM ASPARTIC PROTEINASE INHIBITOR DEVELOPMENT
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    6099924
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PLASMODIUM FALCIPARUM ASPARTIC PROTEINASE INHIBITOR DEVELOPMENT
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    6235343
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    1997
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    $ 4.8万
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PORCINE PEPSINOGEN CRYSTAL STRUCTURE: ASPARTIC PROTEINASE, ZYMOGEN ACTIVATION
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THERMODYNAMICS:PEPSTATIN BINDING TO ASPARTIC PROTEINASE ENDOTHIAPEPSIN:AIDS DRUG
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