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. 2016 May;9(3):355-68.
doi: 10.1111/1751-7915.12350. Epub 2016 Feb 16.

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

Safa Ben Azoun  1 , Aicha Eya Belhaj  1 , Rebecca Göngrich  2 , Brigitte Gasser  2 , Héla Kallel  1
Affiliations

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

Safa Ben Azoun et al. Microb Biotechnol. 2016 May.

Abstract

In 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.

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Figures

Figure 1
Figure 1
RABV‐G protein expression in the selected recombinant Pichia pastoris strains. Copy number of selected recombinant P. pastoris strains of (A) pPICZαA‐RABV‐G and (B) pHIL‐S1‐RABV‐G transformants. Western blot analysis of 15 μl of culture supernatants (concentrated 10‐fold by centricon) of (C) pPICZαA‐RABV‐G, and (D) pHIL‐S1‐RABV‐G transformants. PC (positive control): inactivated and purified rabies virus. NC (negative control): KM71H and GS115His− strains transformed with empty pPICZαA and pHIL‐S1 vectors respectively. The amount of RABV‐G produced by the different clones with α‐factor (E) or PHO1 (F) signal sequence at 72 h of induction as determined by ELISA.
Figure 2
Figure 2
Soluble intracellular RABV‐G protein accumulation in clones with (A) α‐factor and (B) clones with PHO1 signal sequence. Membrane‐associated RABV‐G protein level in clones with (C) α‐factor and (D) in clones with PHO1 signal secretion. Transcription levels of selected UPR‐related genes (PDI1,KAR2,HAC1) in clones with (E) α‐factor and (F) PHO1 as a signal secretion. Transcription levels of ERAD‐related genes (HRD1 and CDC48) in the different Pichia pastoris recombinant strains with α‐factor (G) and (H) PHO1 signal secretion. Gene transcript levels were normalized relative to α‐1 or p‐1 strains containing single copy of RABV‐G gene. α‐2, α‐3, α‐4, α‐7 and α‐8 correspond to selected clones of recombinant P. pastoris strains harbouring different copies of RABV‐G gene and where RABV‐G secretion was driven by the α‐factor. p‐2, p‐3, p‐4, p‐5 and p‐7: selected clones where RABV‐G was directed by PHO1 signal secretion and containing different copies of RABV‐G gene.
Figure 3
Figure 3
Effect of coexpression of five factors involved in oxidative protein folding on the expression level of RABV‐G. Western blot analysis of 15 μl of culture supernatants (concentrated 10‐fold by centricon) of yeast clones with α‐factor or PHO1 sequence leader to direct RABV‐G protein secretion coexpressing (A‐1), PDI1 or ERO1 genes, (B‐1) GPX1 or GLR1 and (C‐1) YAP1 genes. PC (positive control): inactivated and purified rabies virus. NC (negative control): KM71H and GS115His− transformed with an empty pPICZαA and pHIL‐S1 respectively. The numbers 1, 3 and 6 correspond to copy number of different genes contained in each clone. The amount of RABV‐G coexpressed with (A‐2) PDI1 or ERO1, (B‐2) GPX1 or GLR1 and (C‐2) YAP1 produced by the different clones with α‐factor or PHO1 as a signal sequence at 72 h of methanol induction as determined by ELISA. Clone abbreviations are explained in Tables S2, S3 and S4.
Figure 4
Figure 4
Competition of the neutralizing activity of the rabies‐immune serum by recombinant RABV‐G. The neutralizing titres (IU ml−1) were determined in the presence of varying amounts of RABV‐G expressed by α‐7/P6 recombinant clone, NC (negative control): culture supernatant of KM71H strain transformed with empty pPICZαA vector.
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