Purification and characterization of colicin V from Escherichia coli culture supernatants.
The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.
Fath
M J
MJ
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Zhang
L H
LH
Rush
J
J
Kolter
R
R
D000595:Amino Acid Sequence; D015153:Blotting, Western; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003087:Colicins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D014238:Trichloroacetic Acid
D003087:Colicins; D014238:Trichloroacetic Acid