Overexpression of a recombinant gamma-glutamyltranspeptidase from Escherichia coli Novablue.
Indian J Biochem Biophys
; 43(6): 345-50, 2006 Dec.
Article
em En
| MEDLINE
| ID: mdl-17285798
ABSTRACT
A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene, lacking the first 48-bp coding sequence for part of the signal sequence, was amplified by polymerase chain reaction (PCR) and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 21 kDa respectively by SDS-PAGE, indicating the precursor EcGGT still undergoes the post-translational processing even in the truncation of signal sequence. His6-tagged EcGGT migrated relative to the molecular mass of approximately 120 kDa and its heterodimeric structure was confirmed by a native-PAGE gel.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Escherichia coli
/
Gama-Glutamiltransferase
Idioma:
En
Revista:
Indian J Biochem Biophys
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Taiwan