Cloning expression and purification of glycerol dehydrogenase from Klebsiella pneumoniae.
- Author:
Tingting ZHANG
1
;
Baishan FANG
;
Geng WANG
;
Feifei WANG
Author Information
1. Key Laboratory of Industrial Biotechnology Fujian Province, Huaqiao University, Quanzhou 362021, China.
- Publication Type:Journal Article
- MeSH:
Chromatography, Affinity;
Cloning, Molecular;
DNA, Bacterial;
genetics;
Escherichia coli;
genetics;
isolation & purification;
metabolism;
Klebsiella pneumoniae;
enzymology;
genetics;
Sugar Alcohol Dehydrogenases;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2008;24(3):495-499
- CountryChina
- Language:Chinese
-
Abstract:
The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
