Overexpression, Purification, and Characterization of the Thermostable Mevalonate Kinase from Methanococcus jannaschii

Author： Huang K-X. Scott A.I. Bennett G.N.

ISSN： 1046-5928

Source： Protein Expression and Purification, Vol.17, Iss.1, 1999-10, pp. : 33-40

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Abstract

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85°C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15–20% of total soluble protein) when induced with isopropyl β-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 had a dimeric structure composed of identical subunits, and the M_r of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate–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)-mevalonate was 68.5 μM and was 92 μM for ATP. The V_max was 387 units mg^-1. The optimal temperature for mevalonate kinase activity was 70–75°C.