CRISPR knock out of SUBTILISIN protease genes in tobacco BY-2 cells and impact on recombinant protein degradation

Details

Supervisors

Chaumont, François ; Moens, Mathilde

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The N. tabacum cv. BY-2 cell line has several qualities and uses as an expression system for therapeutic recombinant glycoproteins. This system is an adequate host for recombinant glycoprotein expression, yet a major limitation remains which is the degradation of said proteins when secreted. This degradation has been linked to proteolytic activity of extracellular proteases localized in the cell wall and culture medium. Members of the subtilisin-like serine (S8) family were identified in those compartments. The goal of this thesis was to investigate whether this family is implicated in recombinant glycoproteins degradation in BY-2 cells. The CRISPR/Cas9 genome edition system was used to inactivate 16 genes encoding subtilisin-like serine proteases identified in an in-lab mass spectrometry analysis. Homeologous genes were knocked out simultaneously reducing the number of distinct transformations from 16 to 10. Western blot analyses of the KO transformants revealed that degradation of two reporting glycoproteins, an immunoglobulin G type 1 (IgG1) and a viral glycoprotein ectodomain (Vge), remained unchanged. These results seem to exclude that the 16 subtilisin proteases, on their own, are implicated in the reporting glycoprotein degradation.