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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

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.
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mbt212350-fig-0001: 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.

Mentions: For each construction, we selected six recombinant strains and determined the copy number of the integrated expression cassette by real‐time q‐PCR (Fig. 1A). For clones transformed with pPICZαA‐opt‐RABV‐G, two clones named α‐8, harbouring eight copies of RABV‐G gene, and α‐7 containing seven copies were isolated. Clones bearing intermediate number of the expression cassette were also identified, and named α‐3 and α‐4. Clones with low copy were isolated, and designated as α‐1 and α‐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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835572&req=5

mbt212350-fig-0001: 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.
Mentions: For each construction, we selected six recombinant strains and determined the copy number of the integrated expression cassette by real‐time q‐PCR (Fig. 1A). For clones transformed with pPICZαA‐opt‐RABV‐G, two clones named α‐8, harbouring eight copies of RABV‐G gene, and α‐7 containing seven copies were isolated. Clones bearing intermediate number of the expression cassette were also identified, and named α‐3 and α‐4. Clones with low copy were isolated, and designated as α‐1 and α‐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