Cloning, Sequencing, and Expression of the Tulip Bulb Chitinase-1 cDNA

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概要

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• A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a λ ZAP cDNA library with anti-TBC-1 antiserum and the 5' rapid amplification of cDNA end (RACE) method, and sequenced. The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids. Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N-and C-termini, respectively. The deduced sequence of TBC-1 had 10-20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively. The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant TBC-1 (rTBC-1) expressed in E. coli was purified by gel filtration followed by ion-exchange chromatography. Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin. This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.

• 社団法人日本農芸化学会の論文
• 2000-07-23

著者

• YAMAGAMI Takeshi

  Laboratories of Protein Chemistry and Engineering

• Ishiguro Masatsune

  Laboratory Of Protein Chemistry & Engineering Department Of Genetic Resources Technology Facultr

• TSUTSUMI Kazuki

  Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University

• Tsutsumi Kazuki

  Laboratory Of Protein Chemistry And Engineering Faculty Of Agriculture Kyushu University

• Ishiguro Masatsune

  Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University

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