Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter

Abstract

Rabies virus glycoprotein (RABV-G) is responsible for inducing neutralizing antibodies synthesis, which are a key element for the protection against rabies infection. There is an urgent need to develop cost effective vaccines despite the availability of commercial products. In a previous study, we described the expression of RABV-G in the yeast Pichia pastoris under the control of AOX1 promoter and α-factor mating factor from Saccharomyces cerevisiae to target the protein towards secretion. We showed that the expressed RABV-G was recognized by rabies virus neutralizing antibodies; nevertheless the secretion level remained low; being around 128ng/mL. Such a low yield will preclude the use of this system for a biotechnological application. In an attempt to improve the secretion level of RABV-G in Pichia pastoris, we investigated in the current study the impact of the constitutive GAP promoter on the expression of the target protein. The expression level was slightly increased to150ng/mL and the produced RABV-G showed strong antigenicity based on the RFFIT test. Interestingly, co-expression of Pichia pastoris endogenous genes encoding for five factors involved in oxidative protein folding (PDI1, GPX1, ERO1, GLR1 and YAP1) had a beneficial impact on RABV-G expression, which was enhanced to 2261ng/mL.