The preparation and identification of diagnostic recombinant glycerol kinase.
- Author:
Yao MENG
1
;
Zhenwei WANG
;
Shuaikun WANG
;
Jieqing HAO
;
Hui SHI
;
Yanfa MENG
;
Shuangfeng LIN
Author Information
1. School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610083, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
metabolism;
Fermentation;
Glycerol Kinase;
biosynthesis;
genetics;
isolation & purification;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification
- From:
Journal of Biomedical Engineering
2013;30(2):327-337
- CountryChina
- Language:Chinese
-
Abstract:
In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.
