Optimisation of the folding of the human cytomegalovirus glycoprotein B when expressed in plant cells

This master thesis was involved in a global project that aimed to improve the secretion of the HCMV glycoprotein B, a leading vaccine candidate, in the extracellular medium of Nicotiana tabacum BY-2 cell suspension cultures. In order to produce a protein with a high immunogenicity, it has to be in its mono-disperse trimeric conformation with the appropriate N-glycosylation profile. Previous experiments with two different gB isoforms did not meet those requirements. The first gB isoform (gB873) has a mutated furin cleavage site and yielded 20 mg/L of intact gB in the culture medium of BY-2 cells. However, it was produced mainly in aggregates. The second tested isoform (gB759) has, in addition, mutations in the two hydrophobic fusion loops to prevent hydrophobic aggregation. However, gB759 was poorly accumulating in the MS culture medium. In this study, two strategies were implemented to improve the production of gB in BY-2 cells. The first one evaluated the expression of a new HCMV glycoprotein B isoform (gB873Linker) harbouring a flexible 15 amino acid (Gly4Ser)3 linker in place of the furin cleavage site. Both single (gB873Linker) and double (2gB873Linker) expression constructs were used to obtain transgenic BY-2 cell lines. As a result, ELISA assays showed that double construct transformants produced considerably higher amount gB protein. However, Western blotting analyses indicated that a major part of it was present as proteolytic fragments. The second strategy involved the co-expression of the human calreticulin (CRTh), a molecular chaperone involved in the folding of glycoproteins. The impact of CRTh co-expression on gB873Linker production and secretion was evaluated in transgenic BY-2 cells and through transient expression in Nicotiana benthamiana leaves. In transgenic BY-2 cells, production of gB was slightly increased when human CRT was expressed. Furthermore, transient experiments in N. benthamiana leaves showed a clear improvement in gB production suggesting that human CRTs, or at least, CRT levels in plants play an important role in glycoprotein folding.