Abstract
SummaryIn this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV‐G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV‐G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV‐G gene was not tremendously increased as compared with the non‐optimized one. In addition, similar levels of RABV‐G were obtained when α‐factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high‐level expression of RABV‐G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV‐G expression cassette indicating that folding was the limiting step for RABV‐G secretion. To circumvent this limitation, co‐overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1,ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV‐G reached 1230 ng ml−1. Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.
Highlights
The methylotrophic yeast Pichia pastoris (Komagataella sp.) has become a substantial workhorse for biotechnology, especially for heterologous protein production (Kurtzman, 2009; Ahmad et al, 2014)
We report our efforts to enhance the heterologous production of rabies virus glycoprotein (RABV-G) in P. pastoris
The important immunogenic property of RABV-G makes it as an attractive alternative that can be used as a vaccine or as diagnostic antigen in enzyme linked immunosorbent assay (ELISA) for detecting anti-glycoprotein antibodies in immunized host
Summary
The methylotrophic yeast Pichia pastoris (Komagataella sp.) has become a substantial workhorse for biotechnology, especially for heterologous protein production (Kurtzman, 2009; Ahmad et al, 2014) Using this system, a variety of proteins of different origins (human, animal, plant, fungal, bacterial and viral) has been produced with varying degrees of success (Sreekrishna et al, 1997; Leonardo et al, 2012). Several genetic factors can be modified to enhance protein expression such as sequence optimization (Bai et al, 2011), gene copy number (Norden et al, 2011; Shen et al, 2012), promoter selection (Shen et al, 1998; Hohenblum et al, 2004), secretion signal (Koganesawa et al, 2001) and coexpression of folding-assistant proteins (Li et al, 2010; Shen et al, 2012)
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