A novel strategy for the production of fluorescent enterocin CRL35 derivates

MASÍAS, RE; ACUÑA, L; MORERO, RD; MINAHK, CJ

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Bacteriocins are antimicrobial peptides produced by prokaryotes, being of particular interest those synthesized by lactic bacteria, as many of them are considered GRAS (Generally Recognized As Safe). Enterocin CRL35 is a bacteriocin produced by Enterococcus mundtii CRL35, a strain isolated from a regional Argentinean cheese (Tafi del Valle, Tucumán). This antimicrobial peptide was tested as a biopreservative agent on cheese, proving to be effective in controlling the growth of the food-borne pathogen Listeria monocytogenes. As enterocin CRL35 displayed a synergistic effect with conventional antibiotics like erythromycin, chloramphenicol and tetracycline, a potential clinical application was suggested. Since the interaction of this peptide with membranes was not studied in detail, the aim of the present work was to establish a strategy to obtain fluorescent derivatives of enterocin CRL35 in order to analyze the binding to model membrane systems. As a first step, munA (enterocin CRL35 structural gene) was cloned into pET22 vector (Novagen). Then, conditions for induction and production of this antimicrobial peptide were optimized in different strains of Escherichia coli. Enterocin CRL35 was overexpressed in E. coli Rosetta 2 pLysS grown in minimal medium and the peptide was recovered from culture supernatants. Enterocin was purified by ammonium sulfate precipitation followed by reverse phase chromatography (C18-AKTA) and finally, enterocin CRL35 was derivatized with fluorescent probes tetramethylrhodamine N-succinimidyl ester and NBD fluoride. The fluorescent enterocins were purified by gel filtration chromatography and the binding of labeled peptides to liposomes was analyzed with an ISS-PC1 spectrofluorimeter. This strategy turned out to be very useful to obtain enough amounts of antimicrobial peptides for analysis by biophysical methods.