Expression of Nanobodies in Marchantia polymorpha chloroplasts

Abstract

The production of recombinant proteins, such as antibodies and nanobodies, is essential for biomedical and other practical applications. Plants possess many advantages for recombinant protein production, including lower costs, larger scale production compared to other systems and low risk of contamination by mammalian pathogens. Marchantia polymorpha, a liverwort with rapid growth and many experimental tools, presents great potential as a prototyping system to characterise recombinant protein production strategies for plants. In this thesis, I have characterised different approaches for maximising recombinant protein production through stable nuclear expression (Chapter 3) and chloroplast expression (Chapter 4). I then finally attempted to produce an anti-mCherry nanobody tagged with mTurquoise2 (LaM4) through nuclear and chloroplast expression in Marchantia (Chapter 5). For expression of transgenes from the Marchantia nuclear genome, I tested different promoters and subcellular locations of protein products. I found that the cauliflower mosaic virus 35S promoter with dual enhancer (pro35Sx2) and cytoplasmic localisation of the protein product gave the highest yield of recombinant protein: up to ~60µg/g fresh weight (~2% of total soluble protein). However, the growth and accumulation of biomass were negatively impacted in the plants expressing high levels of recombinant protein. For expression from the chloroplast genome, the inclusion of a binding site of a pentatricopeptide repeat (PPR) protein in the 5’ untranslated region (UTR) can boost the yield of recombinant proteins up to ~400µg/g fresh weight (~15% total soluble protein). Yet, this strategy also led to slower growth and decrease of biomass in transgenic plants. For the expression of the LaM4 nanobody, while expression from the chloroplast genome did not yield high yield of the protein, expressing LaM4 from the nuclear genome with a C-terminal 3xFLAG tag resulted in a yield of ~120µg/g fresh weight (~4% of total soluble protein). My work provides a proof of principle that Marchantia can be used for production of useful recombinant protein such as nanobodies.