title: Overproduction and purification of the colicin E1 immunity protein.
abstract: Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector. The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography. A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence. The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes. The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.
authors.affiliation: Department of Biochemistry, Yamaguchi University School of Medicine, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Shirabe
authors.forename: M
authors.initials: M
authors.lastname: Yamada
authors.forename: A R
authors.initials: AR
authors.lastname: Merrill
authors.forename: W A
authors.initials: WA
authors.lastname: Cramer
authors.forename: A
authors.initials: A
authors.lastname: Nakazawa
mesh_terms: D001483:Base Sequence; D003087:Colicins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D007613:Kanamycin Resistance; D008081:Liposomes; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D003087:Colicins; D008081:Liposomes; D011993:Recombinant Fusion Proteins