OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ESCHERICHIA-COLI ACYL CARRIER PROTEIN AND 2 MUTANT PROTEINS

A synthetic gene of 237 bases encoding the 77-residue acyl carrier pro tein (ACP) from Escherichia coli, along with two mutant genes, ACP-I54 V and ACP-A59V, were subcloned into the pET11a-pLysS E. coli overexpre ssion system under the control of the bacteriophage T7 promoter, This efficient expression system and a simplified purification protocol yie lded more than 120 mg/l of pure protein. The construct produced a mixt ure of holo-ACP and apo-ACP and two HPLC procedures were developed to separate the two species, This overexpression system allows cost-effec tive growths of C-13- and N-15-labeled protein for structural and othe r 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 r eversion should be taken. (C) 1995 Academic Press, Inc.