title: Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.
abstract: In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV-G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV-G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one. In addition, similar levels of RABV-G were obtained when α-factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high-level expression of RABV-G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV-G expression cassette indicating that folding was the limiting step for RABV-G secretion. To circumvent this limitation, co-overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV-G reached 1230 ng ml(-1). Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.
authors.affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP. 74, Tunis, 1002, Tunisia.
authors.forename: Safa
authors.initials: S
authors.lastname: Ben Azoun
authors.affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP. 74, Tunis, 1002, Tunisia.
authors.forename: Aicha Eya
authors.initials: AE
authors.lastname: Belhaj
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, 1190, Austria.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Göngrich
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, 1190, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP. 74, Tunis, 1002, Tunisia.
authors.forename: Héla
authors.initials: H
authors.lastname: Kallel
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003062:Codon; D018628:Gene Dosage; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060847:Metabolic Engineering; D009500:Neutralization Tests; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D056811:Unfolded Protein Response; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D057134:Antibodies, Neutralizing; D000956:Antigens, Viral; D003062:Codon; D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus