The preparation and identification of diagnostic recombinant glycerol kinase / 生物医学工程学杂志
Journal of Biomedical Engineering
; (6): 327-337, 2013.
Article
in Zh
| WPRIM
| ID: wpr-234654
Responsible library:
WPRO
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.
Full text:
1
Index:
WPRIM
Main subject:
Recombinant Proteins
/
Escherichia coli
/
Fermentation
/
Genetics
/
Glycerol Kinase
/
Metabolism
Type of study:
Diagnostic_studies
Language:
Zh
Journal:
Journal of Biomedical Engineering
Year:
2013
Type:
Article