Time- and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli.
Protein Expr Purif
; 55(1): 9-16, 2007 Sep.
Article
em En
| MEDLINE
| ID: mdl-17574862
ABSTRACT
The S24F mutant of the chemokine RANTES was found to be partly acetylated when produced in recombinant Escherichia coli BL21(DE3)(pDIA17)(CCL5-S24F-pET-26b). Mass spectrometry and Edman sequencing of peptides generated by lys-C endopeptidase indicated that Lys-26, Lys-34, Lys-46, and Lys-57 were susceptible to acetylation. The extent of acetylation of the RANTES S24F polypeptide increased with temperature and with the time during which the culture was incubated after adding the inducer isopropyl-beta-D-thiogalactoside (IPTG). These findings suggest that induction at low temperature and for a short period of time should be preferred when spurious acetylation is a problem for the production of genuine recombinant polypeptides. Acetylation of the polypeptide was not affected by deleting acs, yfiQ, or speG, which encode acetyl-CoA synthetase, acetyl-CoA synthetase acetylase, and spermidine acetyl transferase, respectively, nor by the presence or absence of the pDIA17 plasmid, which harbours the cat gene encoding chloramphenicol acetyl transferase. By contrast, spontaneous acetylation of RANTES could be demonstrated by incubating either the purified polypeptide or inclusion bodies derived from an induced culture in the presence of acetyl-CoA.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Temperatura Baixa
/
Quimiocina CCL5
/
Escherichia coli
Limite:
Humans
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article