Optimization of the expression and purification of Yersinia enterocolitica OmpC porin

DI GENARO SILVIA; LEPORATI MARIANELA; ELIÇABE, JAVIER

Mendoza

Congreso; XXXVI Reunion Científica de la Sociedad de Biologia de Cuyo; 2016

Yersinia enterocolitica (Ye) are Gram-negativebacteria that cause food borne acute or chronic gastrointestinal diseases. Porinsare abundant proteins found in the outer membrane of Gram-negative bacteria. Porinsform pores that allow the passive transport of nutrients. Moreover, porins havebeen described as activators of the innate and adaptive immune responses. The objective of this study was to optimize the conditionsof high expression and purification of recombinant OmpC. E. coli BL21 (DE3) Star were transformed with pET-OmpC vector and singlecolonies were selected in LB agar containing specific antibiotic. To optimize theconditions of expression, we analyzed various conditions including the IPTGconcentration, induction temperature, glucose addition, induction time and bacterialdensity. We observed that the highest expression of OmpC was obtained at cellulardensity (OD600 nm) of 0.7-1.0. Moreover, we demonstrated that OmpC isnot toxicity to the host cell. Although the addition of 0.1% glucose improved thebacterial growth, this glucose concentration suppressed the basal expression ofOmpC. Moreover, the optimum expression of OmpC started 3 h post-induction with1 mM IPTG at 25°C. However, OmpC was predominantly expressed as insolubleinclusion bodies. The recombinant OmpC was purified by affinity chromatographyand we observed that soluble protein bound to nickel ion-affinity column and waseluted as pure protein with 200 mM imidazole. In conclusion, we have optimizedan efficient protocol to produce high concentrations of recombinant OmpC fromYe in E. coli.