Cloning, sequencing, and expression of the tulip bulb chitinase-1 cDNA.
A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a lambda 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.
Yamagami
T
T
Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Fukuoka, Japan. yamagami@agr.kyushu-u.ac.jp
Tsutsumi
K
K
Ishiguro
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018744:DNA, Plant; D015870:Gene Expression; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002688:Chitinases; C110892:chitinase, class III