Cloning, Expression, and Mutagenesis of Trypsin Inhibitor ETIb from Erythrina variegata Seeds
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概要
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Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not. The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage λgt11. The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 pb from ATG to TGA codons. The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus. The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro^<61> and Phe^<62> in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed. This mutant showed significant tPA inhibitory activity, albeit less than ETIa. The result demonstrates that the Arg^<61> and Leu^<62> residues in ETIa are important in inhibiting tPA, and also suggests that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA.
- 社団法人日本農芸化学会の論文
- 1997-06-23
著者
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YAMASAKI Nobuyuki
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University
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KIMURA Makoto
Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University
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KOUZUMA Yoshiaki
Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University
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Kimura M
Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University
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Yamasaki Nobuyuki
Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University
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Yamasaki Nobuyuki
Laboratory Of Biochemistry And Laboratory Of Protein Chemistry And Engineering Faculty Of Agricultur
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Yamasaki N
Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University
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Kouzuma Yoshiaki
Laboratory Of Food Molecular Functionality College Of Agriculture Ibaraki University
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Kouzuma Yoshiaki
Laboratory Of Food Functionality School Of Agriculture Ibaraki University
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Kawabe Masato
United Graduate School Of Agricultural Science Tokyo University Of Agriculture And Technology (tuat)
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Yamasaki Nobuyuki
Laboratory Of Biochemistry Department Of Bioscience And Biotechnology Graduate School Faculty Of Agr
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Kimura Makoto
Laboratory Of Biochemistry Department Of Bioscience And Biotechnology Graduate School Faculty Of Agr
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Yamasaki Nobuyuki
Laboratory Of Agricultural Process Engineering Faculty Of Agriculture Kyushu University
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Kimura Makoto
Laboratory Of Biochemistry Department Of Agricultural Chemistry Faculty And Graduate School Of Agric
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KOUZUMA Yoshiaki
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University
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KIMURA Makoto
Laboratory for Remediation Research, Plant Science Center, RIKEN
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