Production of intact glycoproteins in Nicotiana tabacum BY-2 cells : Effect of linkers on the integrity of fusion proteins

Details

Supervisors

Chaumont, François ; Navarre, Catherine

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enThe production of recombinant proteins in plant systems offers a promising alternative to traditional expression platforms, particularly for complex glycoproteins. However, proteolytic degradation remains a major challenge. This study investigates the role of linker sequences in improving the stability of fusion proteins expressed in plants. Two model glycoproteins, erythropoietin (EPO) fused to the fluorescent Clover protein, and the receptor-binding domain (RBD) of SARS-CoV-2 fused to a His-tag, were used to evaluate five synthetic linkers: three flexible ([G4S]3, [G]8, [GSAGSAAGSGEF]) and two rigid ([EAAAK]3, [AP]7). These fusion proteins were expressed using transient expression in Nicotiana benthamiana leaves and stable expression in Nicotiana tabacum BY-2 suspension cells. The results revealed a strong context dependence on linker performance. In transient expression, flexible linkers such as [G4S]3 and [G]8 improved RBD stability, while EPO fusion proteins were systematically degraded regardless of the linker. Stable expression in BY-2 cells provided a more favorable environment for analyzing linker efficiency during secretion. The rigid linker [EAAAK]3 conferred significant protection to the EPO-Clover fusion, with detectable levels of intact EPO-Clover in the extracellular medium. The [AP]7 linker exhibited a unique behavior for both constructs, likely due to Hyp-O- glycosylation of proline residues, which enhanced secretion and conferred partial resistance to degradation. These findings highlight the importance of tailoring linker design to both the expression system and the fusion protein. BY-2 cells, with their reduced protease activity and compatibility with optimized media such as D11b, provide a controlled environment for evaluating linker performance. However, some limitations, such as the limited sensitivity of His-probe, small differences in molecular mass between cleaved and intact proteins, and the high susceptibility of EPO to degradation, prevented definitive conclusions from being drawn. Future work should focus on purification, quantitative analysis, and environmental modulation (e.g., pH, co-expression of protease inhibitors) to refine linker selection strategies. This study provides valuable insights into the rational design of fusion proteins for plant molecular farming, thereby supporting the production of intact recombinant glycoproteins.