Expression, purification and characterization of K5 lyase in Escherichia coli.
- Author:
Jinhui GAO
1
;
Wenfang DOU
;
Hui LI
;
Xiaomei ZHANG
;
Hongyu XU
;
Zhenghong XU
Author Information
1. Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Bacterial Capsules;
metabolism;
Escherichia coli;
enzymology;
genetics;
Genetic Vectors;
genetics;
Heparin;
metabolism;
Lyases;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2012;28(6):696-704
- CountryChina
- Language:Chinese
-
Abstract:
K5 polysaccharide of high molecular weight (HLW) can be splitted into low molecular weight (LMW) K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate (HS). To prepare recombinant K5 lyase (Elma) and analyze its biological activity. The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 (DE3). After induction with 0.2 mmol/L IPTG at 16 degrees C for 5 h, Elma was successfully expressed, SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni(2+)-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 degrees C and the optimal reaction pH is 7.0.
