title: Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris.
abstract: Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form.
authors.affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, Tunis, 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, Tunis, Tunisia.
authors.forename: Meriem
authors.initials: M
authors.lastname: Ben Zakour
authors.affiliation: Institut de Recherche, Vétérinaire de Tunisie, Tunis, Tunisia.
authors.forename: Soufien
authors.initials: S
authors.lastname: Sghaier
authors.affiliation: Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Biofermentation Unit, Institut Pasteur de Tunis, Tunis, Tunisia.
authors.forename: Héla
authors.initials: H
authors.lastname: Kallel
mesh_terms: D015870:Gene Expression; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011820:Rabies virus / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins
keywords: ERAD; Pichia pastoris; UPR; cell physiology; gene dosage; rabies virus glycoprotein