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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

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.
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mbt212350-fig-0003: 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.

Mentions: Western blot analysis of culture supernatants of the selected recombinant strains expressing PDI1or ERO1 shows the presence of a single band at the expected size of RABV‐G (Fig. 3A‐1). The intensity of the band depends on the chaperone coexpressed. Coexpression of PDI1 dramatically improved the level of secreted RABV‐G, independent of the secretion signal used; this effect increased with higher gene copy number of PDI1. Insertion of six copies of PDI1 resulted in 9.6‐fold enhancement of secreted RABV‐G when compared with α‐7 and p‐7 strains. Insertion of three or one copy of PDI1 increased the production level 8.6‐fold and 7.9‐fold respectively (Fig. 3A‐2).


Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

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

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.
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835572&req=5

mbt212350-fig-0003: 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.
Mentions: Western blot analysis of culture supernatants of the selected recombinant strains expressing PDI1or ERO1 shows the presence of a single band at the expected size of RABV‐G (Fig. 3A‐1). The intensity of the band depends on the chaperone coexpressed. Coexpression of PDI1 dramatically improved the level of secreted RABV‐G, independent of the secretion signal used; this effect increased with higher gene copy number of PDI1. Insertion of six copies of PDI1 resulted in 9.6‐fold enhancement of secreted RABV‐G when compared with α‐7 and p‐7 strains. Insertion of three or one copy of PDI1 increased the production level 8.6‐fold and 7.9‐fold respectively (Fig. 3A‐2).

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