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

Paper ID Volume ID Publish Year Pages File Format Full-Text
2742 129 2016 9 PDF Available
Title
Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter
Abstract

•The use of GAP promoter enhanced the expression of RABV-G by 1.22 fold compared to AOX1 promoter.•PDI1 and GPX1 are the most important factors involved in RABV-G protein folding.•Coexpression of PDI1 protein improved the expression level of RABV-G by 15-fold.•Recombinant RABV-G was recognized by rabies neutralizing antibodies.

Rabies virus glycoprotein (RABV-G) is responsible for inducing neutralizing antibodies synthesis, which are a key element for the protection against rabies infection. There is an urgent need to develop cost effective vaccines despite the availability of commercial products. In a previous study, we described the expression of RABV-G in the yeast Pichia pastoris under the control of AOX1 promoter and α-factor mating factor from Saccharomyces cerevisiae to target the protein towards secretion. We showed that the expressed RABV-G was recognized by rabies virus neutralizing antibodies; nevertheless the secretion level remained low; being around 128 ng/mL. Such a low yield will preclude the use of this system for a biotechnological application. In an attempt to improve the secretion level of RABV-G in Pichia pastoris, we investigated in the current study the impact of the constitutive GAP promoter on the expression of the target protein. The expression level was slightly increased to150 ng/mL and the produced RABV-G showed strong antigenicity based on the RFFIT test. Interestingly, co-expression of Pichia pastoris endogenous genes encoding for five factors involved in oxidative protein folding (PDI1, GPX1, ERO1, GLR1 and YAP1) had a beneficial impact on RABV-G expression, which was enhanced to 2261 ng/mL.

Keywords
Yeast; Protein production; Optimization; Recombinant DNA; Pichia pastrois; Rabies virus glycoprotein
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Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 113, 15 September 2016, Pages 77–85
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us