Co‐expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor‐β1 into a biologically active protein

Abstract

SummaryTransforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.

Production of complex viral glycoproteins in plants as vaccine immunogens

“…A recent study has suggested that tissue necrosis during heterologous protein expression in planta could be linked to improper cleavage of the signal peptide. The authors reported that substituting the Arabidopsis thaliana chitinase signal peptide for the native signal sequence of transforming growth factor-b1 resulted in high levels of protein production whilst still maintaining a healthy phenotype in N. benthamiana plants (Wilbers et al, 2016). It is also worth noting that the targeting of the recombinant H5 HA protein into the plant secretory pathway resulted in the formation of enveloped VLPs which budded from the plasma membrane in the absence of other accessory proteins (D'Aoust et al, 2008).…”

“…Given these observations, trimeric HIV Env immunogens are often produced in mammalian cell culture by replacing the native cleavage site with a hexa-arginine motif and co-expressing heterologous furin to promote optimal cleavage of the recombinant glycoprotein (Binley et al, 2002). Furin activity does not naturally occur along the plant secretory pathway, although a precedent exists for the co-expression of the protease to produce functional human transforming growth factor b1 in planta, suggesting that theoretically it may be possible to produce cleaved HIV Env glycoproteins in plants (Wilbers et al, 2016). Another potential complication with the production of a proteolytically cleaved glycoprotein is the labile association between the respective subunits.…”

“…We have recently described the development of a platform for the expression of soluble HIV-1 Env gp140 trimers in N. benthamiana plants (patent application: PPA UK 1617480.7). These antigens exploit a 10 residue glycine-rich linker peptide at the interface of the gp120 and gp41 subunits to eliminate the requirement for furin-mediated cleavage, which does not naturally occur in plants (Wilbers et al, 2016). Preliminary immunogenicity studies confirmed that the proteins were immunogenic in rabbits; however, despite eliciting high titres of binding antibodies, the vaccines only induced low levels of neutralizing antibodies.…”

pH Gradient Mitigation in the Leaf Cell Secretory Pathway Alters the Defense Response ofNicotiana benthamianato Agroinfiltration

“…Current efforts to further strengthen the position of plants as valuable protein expression hosts also include the development of metabolic, cellular or phenology engineering approaches to address specific issues related to recombinant protein maturation, stability or recovery. Examples are the ectopic implementation of a dwarf plant phenotype to optimize culture area use in the greenhouse (Nagatoshi et al, 2015), activation of the octadecanoid pathway to reduce ribulose 1,5- bis -phosphate carboxylase oxygenase (Rubisco) loads in leaves prior to protein purification (Robert et al, 2015), or the expression of accessory convertases to generate biologically active forms of therapeutic proteins otherwise requiring chemical refolding after extraction (Wilbers et al, 2016). Other examples are the expression of protease inhibitors to prevent unintended protein degradation by resident proteases (Goulet et al, 2012; Pillay et al, 2014; Jutras et al, 2016), or the expression of an accessory proton channel to stabilize acid-labile and proteolysis-susceptible proteins in the Golgi lumen (Jutras et al, 2015; 2018).…”

Myrtol improves post‑traumatic knee osteoarthritis by regulation of reactive oxygen species, transforming growth factor β1 and apoptosis in a mouse model

“…It participates in the proliferation and differentiation processes of diversified cell types, such as chondrocytes, epithelial cells and fibroblasts, and has essential regulating effects in the developing process of embryo, organs and cartilage. Studies have demonstrated that, in the chondrocytes of rabbits and cattle, TGF-β1 can regulate the synthesis of proteoglycan (4,5). In addition, TGF-β1 has the ability to promote synthesis of type II collagen by reducing consumption of proteoglycan in order to repair the cartilage (6).…”