Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui.
10.19540/j.cnki.cjcmm.20180105.004
- Author:
Bi-Xia WANG
1
;
De-Hong XU
1
;
Chao-Yang TAN
1
;
Ling-Min JIANG
1
;
Yue-Fang LUO
1
;
Lei MENG
1
Author Information
1. Biological Engineering Laboratory, College of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China.
- Publication Type:Journal Article
- Keywords:
Ligustrum quihoui;
bioinformatics analysis;
gene cloning;
glycosyltransferase;
prokaryotic expression
- MeSH:
Cloning, Molecular;
DNA, Complementary;
Glycosyltransferases;
genetics;
Ligustrum;
enzymology;
genetics;
Molecular Docking Simulation;
Phylogeny;
Plant Proteins;
genetics;
Protein Structure, Secondary;
Protein Structure, Tertiary
- From:
China Journal of Chinese Materia Medica
2018;43(4):704-711
- CountryChina
- Language:Chinese
-
Abstract:
According to the previous results from transcriptome analysis of Ligustrum quihoui, a glycosyltransferase gene(xynzUGT) was cloned by rapid amplification of cDNA ends(RACE). The full length cDNA of xynzUGT was 1 598 bp, consisting of 66 bp 5'-UTR, 1 440 bp ORF and 92 bp 3'-UTR. The ORF encoded a 480 amino-acid protein(xynzUGT) with a molecular weight of 54 826.67 Da and isoelectric point of 5.82. The structure of enzyme was analyzed by using bioinformatics method, the results showed that the primary structure contained a highly conserved PSPG box of glycosyltransferase, the secondary structure included α helix(38%), sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face domains(often referred to as a Rossmann domains), between which a substrate binding pocket is sandwiched. The phylogenetic tree analysis indicated that xynzUGT might catalyze glycosylation of phenylpropanoids, such as tyrosol. Further simulation experiment of molecular docking between enzyme and tyrosol showed that Gly138 and Ser285 located in the binding pocket interacted with tyrosol by hydrogen bonding. SDS-PAGE analysis exhibited that the prokaryotic expression system successfully expressed recombinant xynzUGT with molecular weight of 58 370.57 Da, but it exists in the form of non-soluble inclusion bodies. Using the molecular chaperone and enzyme co-expression method, the soluble expression was promoted to some extent. The above works laid the foundation for further studying on enzymatic reaction and clarifying the functional mechanism of enzyme.
