Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii

We report here the first overexpression and characterization of a thermosta ble mevalonate kinase from an archae, Methanococcus jannaschii, a strict an aerobe, which produces methane and grows at pressure of 200 atm and an opti mum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vecto r, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Esc herichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induc ed with isopropyl beta-D-thiogalactopyranoside, The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein h ad a dimeric structure composed of identical subunits, and the M-r of the e nzyme determined by gel chromatography was 68K. Based on sodium dodecyl sul fate-polyacrylamide gel electrophoresis, the subunit M-r was 36,000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K-m) for (RS)-meval onate was 68.5 mu M and was 92 mu M for ATP. The V-max, was 387 units mg(-1 ). The optimal temperature for mevalonate kinase activity was 70-75 degrees C. (C) 1999 Academic Press.