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

Marín Viegas VS, Acevedo GR, Bayardo MP, Chirdo FG, Petruccelli S - Front Plant Sci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP) La Plata, Argentina.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of ELP on the accumulation levels of ER-GFP, sec-RFP and ER-TG2 and vac-TG2. The same amount of total extract was loaded on the gel as can be observed by the amount of RLS stained with Coomassie Brilliant Blue R-250. The immunoblot was developed with anti-GFP, anti-RFP, and anti-TG2 mAb 2G3, for ER-GFP, sec-RFP and ER-TG2, and vac-TG2, respectively. The intensity of the band was quantified by using ImageJ. Three biological independent experiments were performed and each replicate was obtained using leaves from five different plants. Error bars are standard error of the mean (SEM). ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01).
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Figure 3: Effect of ELP on the accumulation levels of ER-GFP, sec-RFP and ER-TG2 and vac-TG2. The same amount of total extract was loaded on the gel as can be observed by the amount of RLS stained with Coomassie Brilliant Blue R-250. The immunoblot was developed with anti-GFP, anti-RFP, and anti-TG2 mAb 2G3, for ER-GFP, sec-RFP and ER-TG2, and vac-TG2, respectively. The intensity of the band was quantified by using ImageJ. Three biological independent experiments were performed and each replicate was obtained using leaves from five different plants. Error bars are standard error of the mean (SEM). ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01).

Mentions: To evaluate the impact of ELP on the accumulation of ER-GFP, sec-RFP, ER-TG2, and vac-TG2 a Western Blot was performed. The same amount of total leaf extracts was load into the gel and as control the amount of RLS in each lane is shown. The intensity of the GFP, RFP, and TG2 bands were quantify as is detailed in Section “Materials and Methods” and the obtained results are shown in Figure 3. For ER-GFP a 2.0-fold increase in the accumulation level was observed by ELP induced PB formation, while not significant differences were found in sec-RFP levels (Figure 3, upper panel). Accumulation levels of ER-TG2 and vac-TG2 were modified from 9,5 ± 1,5 and 9,9 ± 1,4 to 20,9 ± 2,1 and 24,4 ± 2,3 μg/g fresh leaf tissue, respectively, by expression of ELP (Figure 3, lower panel). In conclusion, ELP induced 2.1- and 2.5-fold increase in the accumulation of ER-TG2 and vac-TG2, respectively.


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

Marín Viegas VS, Acevedo GR, Bayardo MP, Chirdo FG, Petruccelli S - Front Plant Sci (2015)

Effect of ELP on the accumulation levels of ER-GFP, sec-RFP and ER-TG2 and vac-TG2. The same amount of total extract was loaded on the gel as can be observed by the amount of RLS stained with Coomassie Brilliant Blue R-250. The immunoblot was developed with anti-GFP, anti-RFP, and anti-TG2 mAb 2G3, for ER-GFP, sec-RFP and ER-TG2, and vac-TG2, respectively. The intensity of the band was quantified by using ImageJ. Three biological independent experiments were performed and each replicate was obtained using leaves from five different plants. Error bars are standard error of the mean (SEM). ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664624&req=5

Figure 3: Effect of ELP on the accumulation levels of ER-GFP, sec-RFP and ER-TG2 and vac-TG2. The same amount of total extract was loaded on the gel as can be observed by the amount of RLS stained with Coomassie Brilliant Blue R-250. The immunoblot was developed with anti-GFP, anti-RFP, and anti-TG2 mAb 2G3, for ER-GFP, sec-RFP and ER-TG2, and vac-TG2, respectively. The intensity of the band was quantified by using ImageJ. Three biological independent experiments were performed and each replicate was obtained using leaves from five different plants. Error bars are standard error of the mean (SEM). ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01).
Mentions: To evaluate the impact of ELP on the accumulation of ER-GFP, sec-RFP, ER-TG2, and vac-TG2 a Western Blot was performed. The same amount of total leaf extracts was load into the gel and as control the amount of RLS in each lane is shown. The intensity of the GFP, RFP, and TG2 bands were quantify as is detailed in Section “Materials and Methods” and the obtained results are shown in Figure 3. For ER-GFP a 2.0-fold increase in the accumulation level was observed by ELP induced PB formation, while not significant differences were found in sec-RFP levels (Figure 3, upper panel). Accumulation levels of ER-TG2 and vac-TG2 were modified from 9,5 ± 1,5 and 9,9 ± 1,4 to 20,9 ± 2,1 and 24,4 ± 2,3 μg/g fresh leaf tissue, respectively, by expression of ELP (Figure 3, lower panel). In conclusion, ELP induced 2.1- and 2.5-fold increase in the accumulation of ER-TG2 and vac-TG2, respectively.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP) La Plata, Argentina.

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.

No MeSH data available.


Related in: MedlinePlus