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

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 a-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 128 ng/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 to 150 ng/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 (PD11, GPX1, ERO1, GLR1 and YAP1) had a beneficial impact on RABV-G expression, which was enhanced to 2261 ng/mL. (C) 2016 Elsevier B.V. All rights reserved.