Cloning and expression of a chitinase gene from Sanguibacter sp. C4.
The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank database. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.
Tao
Yong
Y
Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, Life Science College, Sichuan University, Chengdu 610064, China.
Jin
Hong
H
Long
Zhang-Fu
ZF
Zhang
Li
L
Ding
Xiu-Qiong
XQ
Tao
Ke
K
Liu
Shi-Gui
SG
D000192:Actinomycetales / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012995:Solubility
D011993:Recombinant Fusion Proteins; D002688:Chitinases