Contact Us Add to Favorite
 
NianQi Search Adv Search
33
农业生物技术学报  2019, Vol. 27 Issue (7): 1275-1281    DOI: 10.3969/j.issn.1674-7968.2019.07.015
Articles and Letters Current Issue | Archive | Adv Search |
The Biosynthesis of c-di-GMP Catalyzed by Escherichia coli Dinucleotide Cyclase
JIA Bin, ZHANG Wei, GUO Yu-Jie, KONG Jiang-Nan, YANG Xue-Chen, PANG Bo-Wen, YANG Guo-Yu*, ZHENG Yue-Ting*
College of Animal Husbandry and Medical Engineering, Henan Agricultural University, Zhengzhou 450002, China
Download: PDF (1614 KB)   HTML (1 KB) 
Export: BibTeX | EndNote (RIS)      
Abstract  Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger molecule widely present in bacteria and can participate in innate immune response through stimulator of interferon genes (STING) signaling pathway and is a potential vaccine adjuvant. In order to clarify its innate immune regulation mechanism and evaluate the effect of immunoadjuvant, the efficient synthesis of c-di-GMP needs to be solved urgently. In order to establish a relatively simple and efficient c-di-GMP biosynthesis method to prepare c-di-GMP in vitro, this study constructed Escherichia coli dinucleotide cyclase (DncV) recombinant expression vector, and the target protein DncV was obtained by IPTG (isopropy-β-D-thiogalactoside)-induced expression and His affinity purification; Detection of c-di-GMP from GTP catalyzed by DncV recombinant protein by ultra performance liquid chromatography (UPLC) in vitro. The results showed that the IPTG-inducible expression vector pET-28a-His-DncV was successfully constructed, and the purity of the recombinant protein obtained by affinity purification was 92%. The recombinant protein could generate c-di-GMP in one step by enzymatic reaction; Metal ions had a significant effect on the activity of the enzyme. Three kinds of metal ions (Mg2+, Mn2+, Co2+) could improve the catalytic efficiency, and the other 7 metal ions (Cu2+, Zn2+, Ni2+, Mo2+, Bo2+, Fe2+, Ca2+) inhibited the enzymatic reaction. This study established a method for the synthesis of c-di-GMP by E. coli DncV recombinant protein, which provides basic data for the large-scale preparation and functional study of c-di-GMP.
Key wordsEscherichia coli DncV      c-di-GMP biosynthesis      Recombinant expression      UPLC      Enzymatic reaction     
Received: 14 November 2018     
ZTFLH:  S851.3  
Corresponding Authors: yuetingzheng@126.com; haubiochem@163.com   
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
JIA Bin
ZHANG Wei
GUO Yu-Jie
KONG Jiang-Nan
YANG Xue-Chen
PANG Bo-Wen
YANG Guo-Yu
ZHENG Yue-Ting
Cite this article:   
JIA Bin,ZHANG Wei,GUO Yu-Jie, et al. The Biosynthesis of c-di-GMP Catalyzed by Escherichia coli Dinucleotide Cyclase[J]. 农业生物技术学报, 2019, 27(7): 1275-1281.
URL:  
http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.07.015     OR     http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I7/1275
Copyright © Editorial Board of 农业生物技术学报
Supported by:Beijing Magtech