Cloning, expression, and purification of His-tagged rat mevalonate kinase.
Mevalonate kinase catalyzes the phosphorylation of mevalonic acid to form mevalonate 5-phosphate, which plays a key role in regulating cholesterol biosynthesis in animal cells. Deficiency of mevalonate kinase activity in the human body has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). We cloned the gene of rat mevalonate kinase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5(') of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column in 90% yield to apparent homogeneity. The purified rat mevalonate kinase had a dimeric structure composed of identical subunits. Based on SDS-PAGE, the subunit was 42 kDa. The specific activity of the purified His-tagged rat mevalonate kinase was 32.7 micromol/min/mg and the optimal pH was found to be 7.0-8.0 in phosphate buffer. The Michaelis constant K(M) was 35 microM for (RS)-mevalonate and 953 microM for ATP, respectively. The V(max) was determined to be 38.7 micromol/min/mg. The overexpression of rat mevalonate kinase in E. coli and one-step purification of the highly active rat mevalonate kinase will facilitate further our investigation of this enzyme through site-directed mutagenesis and enzyme-catalyzed reactions with substrate analogs.
Chu
Xiusheng
X
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Li
Ding
D
D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase