Impact of calnexin expression on the production of recombinant glycoproteins in "Nicotiana benthamiana" leaves

Details

Supervisors

Chaumont, François ; Delasalle, Jules

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

enThe production of therapeutic recombinant glycoproteins represents a major research area in the pharmaceutical industry. Over the last decades, different expression systems have been developed to produce various recombinant glycoproteins, such as monoclonal antibodies or viral glycoproteins. Among these systems, plants and plant cells have emerged as promising alternatives. However, this approach, named molecular farming, also comes with its own challenges, such as the relatively low production yield usually observed for complex recombinant N-glycosylated proteins, such as erythropoietin. One possible explanation is the limitation in protein folding capacity, as increased folding demand in the endoplasmic reticulum (ER) can create a bottleneck in the protein folding machinery. The folding of N-glycosylated proteins in the ER is assisted by lectin chaperones, namely calnexin and calreticulin, which interact with N-glycans. Many studies have demonstrated that the co-expression of human calnexin or calreticulin can significantly improve the folding efficiency of N-glycosylated proteins and increase their production. However, it remains unclear whether such improvements require the expression of exogenous calnexin, or if overexpression of endogenous calnexin alone would be sufficient. In this master’s thesis, the impact of different calnexin homologues on the production of recombinant human erythropoietin in "Nicotiana benthamiana" leaves was investigated. Calnexin homologues from "Nicotiana tabacum", "Homo sapiens" and "Chlamydomonas reinhardtii" were evaluated by transient co-expression with human erythropoietin fused to a fluorescent protein. Protein analysis by western blot and fluorescence intensity measurements consistently showed an increased accumulation of human erythropoietin when co-expressed with each of the calnexin homologues. These results suggest a positive impact of elevated calnexin levels on the folding and the production of recombinant human erythropoietin in "N. benthamiana".