Cloning, sequencing, and expression of the tulip bulb chitinase-1 cdna

Takeshi Yamagami, Kazuki Tsutsumi, Masatsune Ishiguro

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1394-1401
Number of pages8
JournalBioscience, Biotechnology and Biochemistry
Volume64
Issue number7
DOIs
Publication statusPublished - 2000

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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