Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.
Protein Expr Purif
; 17(1): 33-40, 1999 Oct.
Article
em En
| MEDLINE
| ID: mdl-10497066
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.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Mathanococcus
/
Fosfotransferases (Aceptor do Grupo Álcool)
Idioma:
En
Revista:
Protein Expr Purif
Ano de publicação:
1999
Tipo de documento:
Article