Cloning, expression, and purification of His-tagged rat mevalonate kinase

Abstract

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 μmol/min/mg and the optimal pH was found to be 7.0–8.0 in phosphate buffer. The Michaelis constant K M was 35 μM for ( RS)-mevalonate and 953 μM for ATP, respectively. The V max was determined to be 38.7 μmol/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.