CLONING AND PURIFICATION OF A HYBRID BACTERIOCIN

ACUÑA, LEONARDO; SESMA, FERNANDO; MORERO, ROBERTO D.; BELLOMIO, AUGUSTO

BIOCELL

INST HISTOL EMBRIOL-CONICET

Año: 2009 p. 97 - 97

0327-9545

Bacteriocins are ribosomally-synthesized antimicrobial peptides produced by Gram (-) and (+) bacteria with antibacterial spectra generally limited to phylogenetically related microbial strains. In order to obtain a broad-spectrum antimicrobial peptide, we carried out a transcriptional fusion between enterocin CRL35 (a bacteriocin produced by Enterococcus mundtii CRL35) and colicin V (a microcin produced by Escherichia coli) structural genes (munA-cvaC). The engineered gene was cloned into the E.coli expression vectors, pET28b+ and pET43.1a, the last one carrying a NusA tag and a factor Xa-cleavage site-encoding sequence upstream from the chimeric gene. Both constructions were expressed into E. coli BL21 (pLysS) after IPTG induction. MunACvaC and NusA-MunA-CvaC resulted in biologically-active fusion proteins. MunA-CvaC was purified by ionic exchange chromatography (SP-sepharose) followed by reversed phase chromatography. NusA-MunA-CvaC was purified by affinity chromatography by means a HiTrap FF Column. To confirm purity and identity, collected active fraction was resolved by SDS-PAGE and revealed with Coomassie stain, western blot (using anti-poly His primary antibody) and antimicrobial activity. The proteolytic cleavage with Factor Xa allowed us to obtain MunA-CvaC protein without extra amino acids in the N-terminal of the molecule