Optimization of the expression and purification of Yersinia enterocolitica OMPC porin

LEPORATI M; CARMONA N; DI GENARO S; ELIÇABE J

Mendoza

Congreso; XXIV Reunión Anual de la Sociedad de Biología de Cuyo (SOBIO); 2016

Sociedad de Biologia de Cuyo

Yersinia enterocolitica (Ye) are Gram-negative bacteria that cause food borne acute or chronic gastrointestinal diseases. Porins areabundant proteins found in the outer membrane of Gram-negative bacteria. Porins form pores that allow the passive transport ofnutrients. Moreover, porins have been described as activators of the innate and adaptive immune responses. The objective of thisstudy was to optimize the conditions of high expression and purification of recombinant OmpC. To OmpC expression,Escherichia coli BL21 (DE3) Star were transformed with pET-OmpC vector and single colonies were selected in LB agarcontaining specific antibiotic. To optimize the conditions of expression, we analyzed various conditions including the IPTGconcentration, induction temperature, glucose addition, induction time and bacterial density. We observed that the highestexpression of OmpC was obtained at cellular density (OD600 nm) of 0.7-1.0. Moreover, we demonstrated that OmpC is nottoxicity to the host cell. Although the addition of 0.1% glucose improved the bacterial growth, this glucose concentrationsuppressed the basal expression of OmpC. Moreover, the optimum expression of OmpC started 3 h post-induction with 1 mMIPTG at 25°C. However, OmpC was predominantly expressed as insoluble inclusion bodies. The recombinant OmpC was purifiedby affinity chromatography and we observed that soluble protein bound to nickel ion-affinity column and was eluted as pureprotein with 200 mM imidazole. In conclusion, we have optimized an efficient protocol to produce recombinant OmpC from Yein E. coli.