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The scFv antibody fragment was expressed in the recombinant yeast Pichia pastoris by using AOX1 promoter, in the presence of methanol, as carbon source and as a model for expression. This antibody is considered as a possible alternative for the diagnosis and treatment of atherosclerosis. An initial study was conducted in the production of the antibody fragment and the growth of yeast with two different work benches, varying the carbon source (glucose and glycerol). The results showed that the maximum cell concentrations, and the best production of antibody, were achieved with maintained working stock with glycerol as carbon source and preservation. Subsequently, a study was conducted in shaker and bioreactor, from a 2 complete factorial design, in which the variables studied were time induction (24, 28 and 32 hours), temperature induction (10, 20 and 30°C), and concentration of the inducer (methanol 0.5, 1.0 and 1.5%) on the response variable production of the antibody fragment. The only variable that had a statistically significant effect, with a confidence level of 95%, was the inducer concentration for the two production systems. The best yields of the antibody fragment were of 27.8 mg/L for the shaker and 41.3 mg/L for the bioreactor. The third study was to assess the production of antibody fragment into the shaker with the yeast at different carbon sources (sucrose, glycerol, glucose and sucrose/glucose 50%). In this study, the ANOVA analysis did not show statistically significant differences. We conducted planning to improve the quantification of antibody fragment in order to decrease the number of steps to avoid losses in the process. After getting a new protocol for the quantification, we carried out a new study to evaluate the production with different carbon sources. The analysis of the results showed that all carbon sources showed statistically significant effects, among which sucrose obtained the highest yield 93.7 mg/L of antibody fragment. These results were compared and analyzed through a microscope, in which with the observations of the yeast showed that the best antibody production was obtained when the yeast had sizes smaller than 0.895 microns. Next, a study was conducted to determine alternative sources of carbon and nitrogen (rice bran and wheat bran) to produce the antibody fragment in place of casein peptone and casaminoacids as substrates. The highest yield obtained in this case was 27.9 mg/L, when casamino was replaced by the wheat bran. Finally, we evaluated the effect of kLa in yeast growth and production of the antibody fragment scFv, the results showed that the best condition for the growth of the yeast was 96.12 h, on the other hand, the best condition for the production antibody fragment was 12.24 h with a final relation yield of 8.12 mg/L antibody/g/L cell.

Produção do fragmento de anticorpo scFv por Pichia pastoris geneticamente modificada