Molecular Cloning of cDNA That Encodes Chymotrypsin Inhibitor ECI from Erythrina variegata Seeds and Its Expression in Escherichia coli

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概要

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• Synthetic oligonucleotides representing all possible sequences of the N-terminal and internal amino acid sequences of the chymotrypsin inhibitor ECI from Erythrina variegata seeds were used to generate a probe specific for ECI-related sequences by the polymerase chain reaction on the E. variegata genomic DNA. A lambda phage cDNA library constructed from poly(A^+) RNA from maturing seeds was screened with the ECI gene thus obtained as a probe and characterized by DNA sequencing. The cloned ECI cDNA comprised 737 nucleotides and one open reading frame that encoded a polypeptide chain of 203 amino acids including a signal peptide composed of 24 amino acids. An expression plasmid was designed for export of the recombinant inhibitor into the periplasm. For this purpose, the cDNA fragment encoding matured ECI was ligated into the NcoI and BamHI sites following the pelB signal sequence in the expression vector pET-22b and expressed in Escherichia coli BL21 (DE3). However, this attempt failed as the recombinant inhibitor caused the formation of inclusion bodies in E. coli cells as a heterologous preprotein (SR-ECI), with the pelB upstream leader. SR-ECI was made soluble and renatured by refolding and reoxidation, and subsequently processed with pronase to give rise to recombinant ECI (R-ECI) that had an extra methionine residue attached to the N-terminal amino acid of ECI. Purified R-ECI inhibited chymotrypsin almost as strongly as authentic ECI.

• 社団法人日本農芸化学会の論文
• 1996-09-23

著者

• YAMASAKI Nobuyuki

  Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University

• KIMURA Makoto

  Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University

• Kimura M

  Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University

• IWANAGA Shiroh

  Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University

• KURAMITSU Jun-ichi

  Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University

• Yamasaki Nobuyuki

  Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University

• Yamasaki Nobuyuki

  Laboratory Of Biochemistry And Laboratory Of Protein Chemistry And Engineering Faculty Of Agricultur

• Yamasaki N

  Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University

• Kuramitsu Jun-ichi

  Laboratory Of Biochemistry Faculty Of Agriculture Kyushu University

• Iwanaga Shiroh

  Laboratory Of Biochemistry Faculty Of Agriculture Graduate School Kyushu University

• Kawabe Masato

  United Graduate School Of Agricultural Science Tokyo University Of Agriculture And Technology (tuat)

• Yamasaki Nobuyuki

  Laboratory Of Biochemistry Department Of Bioscience And Biotechnology Graduate School Faculty Of Agr

• Kimura Makoto

  Laboratory Of Biochemistry Department Of Bioscience And Biotechnology Graduate School Faculty Of Agr

• Yamasaki Nobuyuki

  Laboratory Of Agricultural Process Engineering Faculty Of Agriculture Kyushu University

• Kimura Makoto

  Laboratory Of Biochemistry Department Of Agricultural Chemistry Faculty And Graduate School Of Agric

• KIMURA Makoto

  Laboratory for Remediation Research, Plant Science Center, RIKEN

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