Expression of RTL1, a general siRNA suppressor, to improve production yield in transient and stable plant expression systems

Details

Supervisors

Chaumont, François ; Vandevelde, Romane

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

enPlant molecular farming (PMF) refers to the use of genetically engineered plants to produce recombinant proteins, through the introduction of a transgene encoding the protein of interest. PMF offers many advantages compared to mammalian or microbial platforms, such as their cost-effectiveness, their good manufacturing practices and scalability, their capacity to produce proteins requiring high eukaryotic post-translational modifications, and their low contamination risk. However, PMF generally exhibits lower yields than mammalian cells. One major limitation is RNA silencing, which restricts the expression of recombinant proteins through both mRNA degradation and transcriptional repression. Dicer-like (DCL) proteins are central to RNA silencing as they cleave double-stranded RNAs (dsRNAs), derived from viruses, transgenes, or endogenous RNA precursors, into small interfering RNAs (siRNAs) that mediate sequence-specific gene silencing. Ribonuclease Three-Like 1 (RTL1) is a plant-derived protein involved in plant-virus interactions that cleaves dsRNAs, such as the substrate of DCL2, DCL3 and DCL4, thereby preventing their processing into siRNAs. This master thesis investigated the co-expression of RTL1 with a protein of interest: the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike protein to improve its production. Specifically, RBD was produced in combination with RTL1, Flag-tagged RTL1, a catalytically inactive version of RTL1 (E64A/D71A), P19, and both RTL1 and P19. P19, a viral suppressor of RNA silencing, was used as a benchmark to compare their ability to enhance protein expression. Nicotiana benthamiana leaves and Nicotiana tabacum Bright Yellow 2 (BY-2) suspension cells were used as molecular farming platforms to produce these proteins and study the effectiveness of RTL1 and P19 in enhancing recombinant protein production. Transient expression in N. benthamiana leaves via agroinfiltration showed that RTL1 effectively maintained RBD accumulation from 6 to 8 days post-infiltration (dpi), although it was generally outperformed by P19. However, variability was observed between experiments. At these dpi, RNA silencing was, by deduction, considered a major limitation for recombinant protein production. Co-expression of RTL1 with P19 showed an antagonistic relationship, resulting in reduced RBD accumulation. Stable transformation in BY-2 cells enabled RBD production with RTL1, Flag-tagged RTL1, and the catalytically inactive version of RTL1, although further confirmation is required. Flag-tagged RTL1 repressed RBD accumulation, whereas RTL1 enhanced RBD accumulation in few cell lines.