| Project/Area Number |
62470124
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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 |
製造化学・食品
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ARAI Soichi Faculty of Agriculture, The University of Tokyo, Associate Professor, 農学部, 助教授 (20011934)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Michiko Faculty of Agriculture, The University of Tokyo, Lecturer, 農学部, 助手 (90107409)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1988: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1987: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Keywords | Cysteine proteinase inhibitor / Oryzacystatin / Cloning / パパイン / システインプロティナーゼインヒビター |
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| Research Abstract |
Cysteine proteinases (EC 3.4.22) have a unique characteristic. Papain (EC 3.4.22.2) as a representative cysteine proteinase can even catalyze an aminolysis reaction leading to covalent incorporation of amino acid esters into protein molecules. Using this reaction, we suceeded in incorporating leucine dodecyl ester into probeins for improvement of their functionality. However, it was needed to develop a useful inhibitor which can control such a reaction with papain.
Meanwhile, we found a cysteine proteinase inhibitor in the endosperm of rice seeds. It was a simple polypeptide with an approximate molecular weight of 11,500 that had potent inhibitory activity for papain. Since the inhibitor was homologous with common cystatins of animal origin, we named it as oryzacystatin. We then cloned a full-length cDNA for oryzacystatin by screening with synthesized oligonucleotide probes based on its partial amino acid sequence. Analysis of the nucleotide spquence of the cDNA gave the putative amino acid sequence of oryzacystatin. It contained the consensus sequence QVVAG conserved among most cystatins of animal origin. We also constructed an expression plasmid containing the cDNA at the multicloning site of pUC18 and produced a lacZ' oryzacystatin fusion protein in Escherichia coli. Next, we constructed expression plasmids lacking the 5'- and 3'-regions of the cDNA. It was thus concluded that the N-terminal 21 amino acid residues are not essential for oryzacystatin to elicit its papain-inhibitory activity and also that a portion of the polypeptide segment containing the sequence QVVAG is necessary for efficient elicitation of the inhibitory activity.
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