Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins. Protein Expr Purif 1995 Aug;6(4):394-400
Date
08/01/1995Pubmed ID
8527922DOI
10.1006/prep.1995.1052Scopus ID
2-s2.0-0029346925 (requires institutional sign-in at Scopus site) 38 CitationsAbstract
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.
Author List
Hill RB, MacKenzie KR, Flanagan JM, Cronan JE Jr, Prestegard JHMESH terms used to index this publication - Major topics in bold
Acyl Carrier ProteinBacterial Proteins
Base Sequence
Chromatography, High Pressure Liquid
Cloning, Molecular
DNA Primers
DNA, Bacterial
Escherichia coli
Gene Expression
Genes, Bacterial
Genes, Synthetic
Molecular Sequence Data
Point Mutation
Polymerase Chain Reaction
