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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

Plant purified ER-TG2 and vac-TG2 as antigen for EC diagnosis. (A) Recognition of ER-TG2 and vac-TG2 by three different monoclonal antibodies (mAb). (B) ELISA with IgA sera of celiac patient and normal healthy donors. ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01). (C) Immunoblot with a pool of IgA sera of celiac patient (Pool+) and normal healthy donors (Pool–). The protein loaded on the gel: ER-TG2, vac-TG2, caco extract is indicated at the top of the immunoblot.
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Figure 4: Plant purified ER-TG2 and vac-TG2 as antigen for EC diagnosis. (A) Recognition of ER-TG2 and vac-TG2 by three different monoclonal antibodies (mAb). (B) ELISA with IgA sera of celiac patient and normal healthy donors. ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01). (C) Immunoblot with a pool of IgA sera of celiac patient (Pool+) and normal healthy donors (Pool–). The protein loaded on the gel: ER-TG2, vac-TG2, caco extract is indicated at the top of the immunoblot.

Mentions: Endoplasmic reticulum-transglutaminase 2 and vac-TG2 were purified from leaves using immobilized metal ion affinity chromatography. To test their usefulness as antigen their recognition by the mAbs 2G3, 5G7, or 4E1, which recognize different TG2 epitopes, was analyzed by immunoblot. Both vac-TG2 and ER-TG2 were positively recognized by these antibodies as is shown Figure 4A, confirming that although in humans TG2 is a localized in the cytosol, the introduction into the plant secretory pathway do not affect the structure of the epitopes recognized by these mAbs. In order to test the performance of the plant purified ER-TG2 and vac-TG2 version in CD screening test an ELISA was performed using a pool of 12 sera of CD patient and control healthy donors (Figure 4B). We found that the pool of CD sera recognized both ER-TG2 and vac-TG2 with a large significant difference over the value obtained for the healthy donors. Plant purified TG2 recognition was also assayed by Western Blot (Figure 4C) confirming that the full-length ER- and vac-TG2 variants were recognized by CD sera while not recognition occurred for control sera. Therefore the plant-produced ER- and vac-TG2 versions conserved the epitopes recognized by IgA sera of celiac individuals. These results point out the usefulness of plant-produced TG2 for develop CD screening tests.


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)

Plant purified ER-TG2 and vac-TG2 as antigen for EC diagnosis. (A) Recognition of ER-TG2 and vac-TG2 by three different monoclonal antibodies (mAb). (B) ELISA with IgA sera of celiac patient and normal healthy donors. ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01). (C) Immunoblot with a pool of IgA sera of celiac patient (Pool+) and normal healthy donors (Pool–). The protein loaded on the gel: ER-TG2, vac-TG2, caco extract is indicated at the top of the immunoblot.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Plant purified ER-TG2 and vac-TG2 as antigen for EC diagnosis. (A) Recognition of ER-TG2 and vac-TG2 by three different monoclonal antibodies (mAb). (B) ELISA with IgA sera of celiac patient and normal healthy donors. ∗∗Denotes statistically significant difference by Student’s t-test (P < 0.01). (C) Immunoblot with a pool of IgA sera of celiac patient (Pool+) and normal healthy donors (Pool–). The protein loaded on the gel: ER-TG2, vac-TG2, caco extract is indicated at the top of the immunoblot.
Mentions: Endoplasmic reticulum-transglutaminase 2 and vac-TG2 were purified from leaves using immobilized metal ion affinity chromatography. To test their usefulness as antigen their recognition by the mAbs 2G3, 5G7, or 4E1, which recognize different TG2 epitopes, was analyzed by immunoblot. Both vac-TG2 and ER-TG2 were positively recognized by these antibodies as is shown Figure 4A, confirming that although in humans TG2 is a localized in the cytosol, the introduction into the plant secretory pathway do not affect the structure of the epitopes recognized by these mAbs. In order to test the performance of the plant purified ER-TG2 and vac-TG2 version in CD screening test an ELISA was performed using a pool of 12 sera of CD patient and control healthy donors (Figure 4B). We found that the pool of CD sera recognized both ER-TG2 and vac-TG2 with a large significant difference over the value obtained for the healthy donors. Plant purified TG2 recognition was also assayed by Western Blot (Figure 4C) confirming that the full-length ER- and vac-TG2 variants were recognized by CD sera while not recognition occurred for control sera. Therefore the plant-produced ER- and vac-TG2 versions conserved the epitopes recognized by IgA sera of celiac individuals. These results point out the usefulness of plant-produced TG2 for develop CD screening tests.

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