Time- and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli.
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.
d'Alayer
Jacques
J
Plate-forme d'Analyse et de Microséquençage des Protéines, Institut Pasteur, 25-28, rue du Dr Roux, 75724 Paris Cedex 15, France.
Expert-Bezançon
Nicole
N
Béguin
Pierre
P
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics; D000595:Amino Acid Sequence; D018946:Chemokine CCL5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018925:Chemokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013696:Temperature; D013997:Time Factors
D018946:Chemokine CCL5; D018925:Chemokines; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C561243:acetyl-CoA synthetase acetylase, E coli; D008239:Lysine