title: [The preparation and identification of diagnostic recombinant glycerol kinase].
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.
authors.affiliation: School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610083, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Meng
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Wang
authors.forename: Shuaikun
authors.initials: S
authors.lastname: Wang
authors.forename: Jieqing
authors.initials: J
authors.lastname: Hao
authors.forename: Hui
authors.initials: H
authors.lastname: Shi
authors.forename: Yanfa
authors.initials: Y
authors.lastname: Meng
authors.forename: Shuangfeng
authors.initials: S
authors.lastname: Lin
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005991:Glycerol Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D005991:Glycerol Kinase