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Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

Ben Azoun S, Belhaj AE, Göngrich R, Gasser B, Kallel H - Microb Biotechnol (2016)

Bottom Line: Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one.Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression.Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP. 74, Tunis, 1002, Tunisia.

No MeSH data available.


Related in: MedlinePlus

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.
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mbt212350-fig-0002: 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.

Mentions: To check if RABV‐G protein was retained within the cell, cell extracts were divided into soluble and membrane‐associated fractions (including the secretory organelles) according to Hohenblum et al. (2004) and analysed by Western blot (Fig. 2). The intensity of bands obtained by different clones was more pronounced in the soluble fraction than in the membrane‐associated fraction for clones utilizing either α‐factor or PHO1 as a secretion signal. Figure 2 shows that the gene copy number impacts the number and the intensity of the intracellular product bands. High copy strains such as α‐8 and α‐7 showed more intense bands than intermediate copy strains (α‐4 and α‐3). By contrast, for low copy strains (α‐1 and α‐2), minor level of RABV‐G was seen in the soluble fraction (Fig. 2). In addition, for high copy strains (as α‐8 and α‐7), a band with a molecular weight lower than 66 kDa was observed. This band could be due to degradation of the protein of interest. For clones where the secretion of RABV‐G protein was directed by PHO1 signal, the accumulation of full length RABV‐G protein in the soluble fraction was lower than that seen for α‐factor clones (Fig. 2). Nevertheless, the degradation of RABV‐G protein was more prominent even for the low copy strains. However for α‐factor clones, the proportion of RABV‐G retained in the membrane‐associated fraction was more prominent than that obtained for PHO1‐strains. This effect was prominent for the high copy strains (Fig. 2C and D).


Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

Ben Azoun S, Belhaj AE, Göngrich R, Gasser B, Kallel H - Microb Biotechnol (2016)

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.
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mbt212350-fig-0002: 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.
Mentions: To check if RABV‐G protein was retained within the cell, cell extracts were divided into soluble and membrane‐associated fractions (including the secretory organelles) according to Hohenblum et al. (2004) and analysed by Western blot (Fig. 2). The intensity of bands obtained by different clones was more pronounced in the soluble fraction than in the membrane‐associated fraction for clones utilizing either α‐factor or PHO1 as a secretion signal. Figure 2 shows that the gene copy number impacts the number and the intensity of the intracellular product bands. High copy strains such as α‐8 and α‐7 showed more intense bands than intermediate copy strains (α‐4 and α‐3). By contrast, for low copy strains (α‐1 and α‐2), minor level of RABV‐G was seen in the soluble fraction (Fig. 2). In addition, for high copy strains (as α‐8 and α‐7), a band with a molecular weight lower than 66 kDa was observed. This band could be due to degradation of the protein of interest. For clones where the secretion of RABV‐G protein was directed by PHO1 signal, the accumulation of full length RABV‐G protein in the soluble fraction was lower than that seen for α‐factor clones (Fig. 2). Nevertheless, the degradation of RABV‐G protein was more prominent even for the low copy strains. However for α‐factor clones, the proportion of RABV‐G retained in the membrane‐associated fraction was more prominent than that obtained for PHO1‐strains. This effect was prominent for the high copy strains (Fig. 2C and D).

Bottom Line: Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one.Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression.Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP. 74, Tunis, 1002, Tunisia.

No MeSH data available.


Related in: MedlinePlus