PURIFICATION AND CHARACTERIZATION OF COLICIN-V FROM ESCHERICHIA-COLI CULTURE SUPERNATANTS

The peptide antibiotic, colicin V (ColV), has been purified and charac terized from Escherichia coil culture supernatants by precipitation wi th trichloroacetic acid (TCA) and high-performance liquid chromatograp hy (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analy sis identifies ColV as a polypeptide with an apparent molecular mass o f 5.8 kDa. The protein identified remains biologically active after pu rification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the ca rboxy-terminal 21 amino acids and replaces them with eight heterologou s 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/Tol C. 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 seque nce 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 pe diocins from Gram-positive bacteria. Mass spectroscopy of purified Col V shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme prov ides a rapid and simple way to obtain ColV for further biochemical ana lysis.