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

Protein Expr Purif. 1999 Oct;17(1):33-40. doi: 10.1006/prep.1999.1106.

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 degrees 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 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 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 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Conserved Sequence
  • DNA Primers / genetics
  • Dimerization
  • Enzyme Stability
  • Escherichia coli / genetics
  • Gene Expression
  • Genes, Archaeal
  • Genetic Vectors
  • Hot Temperature
  • Isoelectric Point
  • Kinetics
  • Methanococcus / enzymology*
  • Methanococcus / genetics*
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphotransferases (Alcohol Group Acceptor) / genetics*
  • Phosphotransferases (Alcohol Group Acceptor) / isolation & purification*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Protein Structure, Quaternary
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • DNA Primers
  • Recombinant Proteins
  • Phosphotransferases (Alcohol Group Acceptor)
  • mevalonate kinase