Expression analysis of glycosyltransferase BcUGT1 from Bupleurum chinense DC. and its expression in E. coli and the target protein purification.
- Author:
Yun-Wen TAO
1
;
Jie-Sen XU
;
Jian-He WEI
;
Jing SUN
;
Yan-Hong XU
;
Xin YANG
;
Yan ZHANG
;
Juan LIU
;
Chun SUI
Author Information
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Base Sequence;
Bupleurum;
chemistry;
Cloning, Molecular;
DNA, Complementary;
genetics;
DNA, Plant;
genetics;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
Glycosyltransferases;
genetics;
isolation & purification;
metabolism;
Oleanolic Acid;
analogs & derivatives;
biosynthesis;
Open Reading Frames;
genetics;
Phylogeny;
Plants, Medicinal;
chemistry;
Protein Structure, Secondary;
Recombinant Fusion Proteins;
genetics;
metabolism;
Saponins;
biosynthesis
- From:
Acta Pharmaceutica Sinica
2013;48(8):1345-1352
- CountryChina
- Language:Chinese
-
Abstract:
The ORF sequence of glycosyltransferase gene BcUGT1 cloned from Bupleurum chinense DC. was analyzed and its three dimentional structure was predicted. Using qRT-PCR method, the expression characteristics of BcUGT1 after methyl jasmonate (MeJA) induction and in different plant tissues were investigated. The results showed that BcUGT1 may be involved in saikosaponin biosynthesis in B. chinense. Thereafter, the recombinant vectors of BcUGT1 were constructed for its expression in E. coli. The target protein was successfully expressed and purified. In the present study, three vectors, pRSET-A, pET-28a (+) and pET-30a (+), and three isolates of E. coli, BL21 (DE3) plysS, BL21A1 and BL21-CodonPlus (DE3)-RIPL were used under different induction conditions, such as different concentrations and during times of inducers (L-arabinose and IPTG) and different inducing temperatures. The results showed that in the condition of 0.5 or 1 mmol x L(-1) IPTG, 16 degrees C, 20 h, target protein expressed in BL21-CodonPlus (DE3)-RIPL with pET-28a (+) or pET-30a (+) as vector. Using PrepEase His-tagged protein purification kit, the target protein was purified. The present work will be helpful for follow-up bio-function analysis of BcUGT1.
