Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter

Azoun, Safa Ben; Belhaj, Aicha Eya; Kallel, Héla

doi:10.1016/j.bej.2016.05.013

Cited by 10 publications

(8 citation statements)

References 39 publications

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“…(), they showed that the genes involved in dissimilatory pathway were upregulated in high copy clones expressing porcine insulin precursor (PIP) under the control of AOX1 promoter. The significant activation of methanol assimilation pathway compared to central carbon metabolism pathway could be the reason for reduced RABV‐G protein production in the aox7 clone compared to gap7 clone, particularly when we take into account that RABV‐G transcript level was similar for both clones (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ). These data indicate that methanol does not offer enough carbon and energy to strongly sustain cell growth and RABV‐G production.…”

Section: Discussionmentioning

confidence: 99%

“…Expression levels of RABV‐G obtained by these clones were compared and a significant difference was seen in extracellular and intracellular compartments. We showed that this difference was obviously substrate and not promoter‐dependent as mRNA levels of RABV‐G gene were comparable (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ).…”

Section: Introductionmentioning

confidence: 87%

“…Optimized RABV‐G gene (Genbank accession number KT878717 ) was used for the construction of the expression cassette. The generation of multi‐copy clones used in this work (gap7, aox7) was previously described in details (Ben Azoun, Belhaj, Göngrich, et al., ; Ben Azoun, Belhaj, & Kallel, ); these clones were constructed using two different recombinant vectors pPICZαA or pGAPZαB. The expression was controlled by AOX1 or GAP as a promoter, α‐mating factor of S. cerevisiae was used to direct the recombinant protein to extracellular medium.…”

Section: Methodsmentioning

confidence: 99%

“…In previous studies, we generated two recombinant clones of P. pastoris KM71H Mut S harboring seven copies of the rabies virus glycoprotein (RABV‐G) gene (Ben Azoun, Belhaj, Göngrich, Gasser, & Kallel, ; Ben Azoun, Belhaj, & Kallel, ). The expression of the target protein was driven either by AOX1 promoter (aox7) or GAP promoter (gap7) and directed in both clones to secretion by the alpha mating factor of Saccharomyces cerevisiae .…”

Section: Introductionmentioning

confidence: 99%

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Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach

Azoun

Kallel

2017

MicrobiologyOpen

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Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV‐G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 ( AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV‐G in P. pastoris.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach

Azoun

Kallel

2017

MicrobiologyOpen

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“…In a previous study, we found an HBsAg-binding protein that could promote the immune process both in vitro and in vivo. Its adjuvanticity in connection with several antigens (HBsAg, rabies [16], and rotavirus [17]) has been evaluated in mice and the results indicate that it has the potential to be a new adjuvant for use in human vaccines.…”

Section: Discussionmentioning

confidence: 99%

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine

Wang

Liu

et al. 2017

PLoS ONE

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Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding protein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vaccine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant.

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Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

Adivitiya

Babbal

Mohanty

et al. 2018

Appl Microbiol Biotechnol

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Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter

Cited by 10 publications

References 39 publications

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

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