Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

Abstract

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

“…Here, we showed that a mouse IgG accumulated at increased yields in leaf central vacuole indicating low‐Ab‐related proteolytic activities in this compartment. Similar results were reported for human α‐mannosidase (De Marchis et al ., ), human complement factor C5a, (Nausch et al ., ), collagen (Stein et al ., ), human α1‐proteinase inhibitor (Jha et al ., ) and human transglutaminase 2 (Marin Viegas et al ., ).…”

“…Retention of Abs in the endoplasmic reticulum (ER) frequently results in increased yields (De Muynck et al ., ). Although the plant central vacuole is considered a hostile environment for foreign protein accumulation, it has been shown that some proteins, such as human alpha‐mannosidase (De Marchis et al ., ), human complement factor C5a, (Nausch et al ., ), collagen (Stein et al ., ) and human transglutaminase 2 (Marin Viegas et al ., ), accumulate in this organelle. In contrast, in carrot suspension cell cultures, secretory versions of human IgG1 and G4 have higher yields than ER and vacuolar variants (Shaaltiel et al ., ).…”

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

“…tumefaciens transient expression system to accelerate the development of a malaria vaccine candidate, with screens for expression, solubility, and stability using fluorescent fusion proteins. In Marin Viegas et al [60], a transient expression system for the production of human TG2 in N. benthamiana leaves was optimized, and the reactivity of plant-produced TG2 in a CD screening test was evaluated. Hence, transient expression performed in contained facilities satisfies good manufacturing practices, and quick expression can avoid the time-consuming stable transformation [58,61].…”

Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

“…The type of cell compartment in which they are found directly influences the folding, assembling, and posttranslational modifications processes, as well as preventing immediate degradation and interference of the polypeptides with cell metabolism (Ahmad et al, 2010;Leite et al, 2019). Typically, the main compartments to which recombinant proteins are targeted include the apoplast, the vacuole, the endoplasmic reticulum, the chloroplasts, and the cytosol (Pereira el at., 2014;Viegas et al, 2015;Miletic et al, 2016) It has also been shown that adding fusion tags, such as elastinlike polypeptides (ELP), hydrophobins (HFBI), and Zera (an Nproline-rich region of gamma zein) to recombinant proteins increases their levels of accumulation. In general, ELP fusions increased the production yield of these target proteins by 2-to 100-fold (Conley et al, 2011) and HFBI fusion led to an increase of GFP accumulation in Nicotiana benthamiana leaves from 18% to 38% of TSP (Joensuu et al, 2010).…”

“…The results obtained using the subcellular targeting strategies or fusion to insoluble tags depend on the nature of the recombinant protein (Viegas et al, 2015). Although other recombinant DENV proteins have been previously targeted to the ER (Seajung et al, 2007;Kim et al, 2009), these strategies have not been tested on the DENV NS1 accumulation in plants.…”