Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins.
A synthetic gene of 237 bases encoding the 77-residue acyl carrier protein (ACP) from Escherichia coli, along with two mutant genes, ACP-I54V and ACP-A59V, were subcloned into the pET11a-pLysS E. coli overexpression system under the control of the bacteriophage T7 promoter. This efficient expression system and a simplified purification protocol yielded more than 120 mg/l of pure protein. The construct produced a mixture of holo-ACP and apo-ACP and two HPLC procedures were developed to separate the two species. This overexpression system allows cost-effective growths of 13C- and 15N-labeled protein for structural and other studies on ACP. In the course of the work on the mutants of ACP, an apparent homologous recombination event led, in one case, to reversion to a wild-type protein, suggesting that precautions to prevent such reversion should be taken.
Hill
R B
RB
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
MacKenzie
K R
KR
Flanagan
J M
JM
Cronan
J E
JE
Prestegard
J H
JH
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial