Expression and purification of a recombinant S-Type bacteriocin in Escherichia coli.

GODINO, A.; LOPEZ-RAMIREZ, V.; FERNANDEZ, M.; FISCHER, S.; BARRA, J. L.

Congreso; LVI Annual SAIB Meeting and SAMIGE; 2020

SAIB

Bacteriocins are proteinaceous antimicrobials with antagonist activity against bacteria phylogenetically related to the producing strain and the interest in them lies in their potential for application in the food industry, medicine, and agriculture. Rhizospheric strain Pseudomonas fluorescens SF39a produces an S-type bacteriocin which inhibits phytopathogenic strains of the genera Pseudomonas and Xanthomonas. These bacteriocins are released into the medium as a binary complex consisting of a larger protein with antimicrobial activity and a smaller immunity protein that remains tightly bound to the cytotoxic domain of the former. In an earlier experiment, a mutant strain SF39a was constructed in the pys gene that encodes the S-type bacteriocin. The mutant presented a deficient phenotype in bacteriocin production compared to the wild type strain, which indicates that the S-type bacteriocin produced by SF39a is functional. The present work aimed to overexpress this bacteriocin as a recombinant protein, using E. coli as the expression system. For this, primers were designed to amplify the bacteriocin and its immunity gene from P. fluorescens SF39a. The PCR product was purified and cloned into the expression vector pET15b(+). This genic construction enabled the production of the S-type bacteriocin fused to an N-terminal His-tag. The expression vector was transformed into strain E. coli BL21 (DE3) pLysS. The recombinant strain was incubated in LB medium with ampicillin and chloramphenicol at 37°C until OD of 0.6. After incubation, the culture was induced with 0.4 mM IPTG or 0.17% lactose overnight at 20°C. Cells were harvested and lysed. Then cell lysates were clarified by centrifugation to obtain the soluble protein fraction. The soluble protein extracts were analyzed by SDS-PAGE. The bacteriocin was overexpressed with both inducers. Given that the expression levels obtained with lactose were similar to those obtained with IPTG, lactose could be used as a replacement for IPTG. Subsequently, the His-tagged bacteriocins were purified from the soluble protein extract using immobilized metal affinity chromatography. For this, the supernatant was incubated overnight with Ni-NTA Agarose Resin at 4°C. After exhaustively washing the column, the protein was eluted using an imidazole gradient. Finally, thepurified protein samples were desalted and analyzed by SDS-PAGE. The results show that S-type bacteriocin from SF39a can be successfully overexpressed in E. coli and recovered with high purity. This methodology would make it possible to produce a greater number of bacteriocins in a simple and economical way. Further studies are necessary to characterize the functionality of the recombinant bacteriocin produced.