High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli.
Gene
; 160(2): 263-7, 1995 Jul 28.
Article
em En
| MEDLINE
| ID: mdl-7642107
ABSTRACT
Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful. To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5'-end of the cDNA was altered to accommodate E. coli codon usage without altering the amino-acid coding sequence. The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications. Following overnight induction of the E. coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins. IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oxirredutases
/
Proteínas Recombinantes de Fusão
/
Oxirredutases atuantes sobre Doadores de Grupo CH-CH
Limite:
Humans
Idioma:
En
Revista:
Gene
Ano de publicação:
1995
Tipo de documento:
Article
País de afiliação:
Estados Unidos