Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana.
- Author:
Yaping QU
1
;
Zhijun ZHANG
1
;
Chaoli WANG
1
;
Lei WANG
1
;
Linjun WU
1
Author Information
- Publication Type:Journal Article
- Keywords: Arabidopsis thaliana; MALDI-TOF MASS; arabinose-5-phosphate isomerase; enzyme properties; heterologous expression; purification
- MeSH: Aldose-Ketose Isomerases; biosynthesis; Arabidopsis; enzymology; Arabidopsis Proteins; biosynthesis; Cloning, Molecular; Escherichia coli; metabolism; Metals; Pentosephosphates; Recombinant Proteins; biosynthesis
- From: Chinese Journal of Biotechnology 2016;32(8):1060-1069
- CountryChina
- Language:Chinese
- Abstract: Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
