Expression of Hepatitis B Surface Antigen major genotypes in South America (F4 and F1b) in Pichia pastoris and purification for in vitro diagnosis

LIMERES MJ; TOSCANINI MA; GOMEZ ER; NUSBLAT AD; CUESTAS ML

Congreso; Reunión conjunta de Sociedades de Biociencias.; 2017

It has been shown that the immune anti-Hepatitis B virus surface antigen (HBs) response to genotype F could be better detected by homologous genotype antigens. As a consequence, we could assess that the use of different antigen genotypes in diagnosis could support a better detection of anti-HBs antibodies against the major genotypes in South America. In order to achieve this goal, we describe in this study the expression in Pichia pastoris and the purification of the HBsAg corresponding to the S region of (sub) genotypes A, F1b and F4.These immunogens were expressed in the methylotrophic yeast P. pastoris, as virus-like particles and cultivated to high cell density in Erlenmeyer flasks as well as in a stirred-tank bioreactor. Expression was induced with methanol pulses and cultures have been set up for 72h. The cells were then lysed by mechanical disruption and the recombinant proteins were purified first by adsorption-desorption on Aerosil-380 followed by ultracentrifugation on discontinuous sucrose gradient, dialysis to remove sucrose and finally concentration with AMICON 10K centrifuge tubes. Purified proteins were analyzed by Coomassie and silver-stained SDS-PAGE gels and its antigenicity was demonstrated by chemiluminescent microparticle immunoassay (CMIA).All recombinant proteins have been efficiently produced in bioreactor with high yield and purified using a short original process in comparison with the growth obtained in Erlenmeyer flasks. The antigenicity of the HBsAg from the different (sub) genotypes was measured by CMIA. Results demonstrated that the different (sub) genotypes have reacted differentially in the CMIA assays. The different HBsAg (sub) genotypes expressed in P. pastoris are going to be used in a novel immunoassay to assess the usefulness of mixing different genotypes.