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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

Subcellular targeting strategies tested for stabilize TG2 in leaves. (A) Schematic representation of the TG2 constructs used for Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. Cyto-TG2 is a cytosolic form of TG2. Sec-TG2, ER-TG2, and Vac-TG2 are introduced in the secretory pathway with murine signal peptide (SP) from gamma 1 antibody chain; SEKDEL, ER retention SP; KISIA is a CT vacuolar targeting signal of the amaranth 11S globulin. Scheme is not drawn to scale. (B) Enzyme-linked Immunosorbent Assay (ELISA) of TG2 fused to the different sorting signals. Microwells were coated with the same amount of total leaves extract overnight at 4°C. After blocking, anti-TG2 mAb 2G3 was added, followed of incubation with a biotin-conjugated anti-mouse, later with HRP-conjugated streptavidin and developed with TMB peroxidase substrate. Three biological replicates (each replicate containing five leaf disks of the infiltrated tissue from a different plant) were used for ELISA. Error bars represent the standard error of the mean (SEM). ∗∗∗∗Denotes statistically significant difference by Tukey’s multiple comparisons test (P < 0.001). (C) Western blot of TG2 fused to the different sorting signals. Expression levels were measured by scanning densitometry of Western Blot developed with 2G3 mAb with a minimum of three independent experiments. The amount of total extract was adjusted using RLS stained with Coomassie Brilliant Blue R-250 as loading control.
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Figure 1: Subcellular targeting strategies tested for stabilize TG2 in leaves. (A) Schematic representation of the TG2 constructs used for Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. Cyto-TG2 is a cytosolic form of TG2. Sec-TG2, ER-TG2, and Vac-TG2 are introduced in the secretory pathway with murine signal peptide (SP) from gamma 1 antibody chain; SEKDEL, ER retention SP; KISIA is a CT vacuolar targeting signal of the amaranth 11S globulin. Scheme is not drawn to scale. (B) Enzyme-linked Immunosorbent Assay (ELISA) of TG2 fused to the different sorting signals. Microwells were coated with the same amount of total leaves extract overnight at 4°C. After blocking, anti-TG2 mAb 2G3 was added, followed of incubation with a biotin-conjugated anti-mouse, later with HRP-conjugated streptavidin and developed with TMB peroxidase substrate. Three biological replicates (each replicate containing five leaf disks of the infiltrated tissue from a different plant) were used for ELISA. Error bars represent the standard error of the mean (SEM). ∗∗∗∗Denotes statistically significant difference by Tukey’s multiple comparisons test (P < 0.001). (C) Western blot of TG2 fused to the different sorting signals. Expression levels were measured by scanning densitometry of Western Blot developed with 2G3 mAb with a minimum of three independent experiments. The amount of total extract was adjusted using RLS stained with Coomassie Brilliant Blue R-250 as loading control.

Mentions: Although TG2 from Caco2 cell line was cloned in E. coli expression vector and different conditions were assayed to produce it, low recovery levels were obtained. For that reason in this work, we attempted to produce TG2 in plant cells. To this end, four versions of TG2 in plant expression binary vector were obtained: cytosolic (cyto-TG2), secretory (sec-TG2), ER-TG2, and vacuolar (vac-TG2), and their schematic representations are shown in Figure 1A. For vacuolar sorting the C terminal KISIA sequence from the amaranth 11S storage globulin was added to TG2 (Petruccelli et al., 2007). To facilitate purification a six histidine tag was also fused to TG2 (Figure 1A). The four TG2 constructs were introduced in A. tumefaciens GV3101 and leaves of N. benthamiana were infiltrated with these agrobacteria. Five days after infiltration, leaves were collected and accumulation of TG2 was measure by ELISA using anti-TG2 mAb 2G3. Figure 1B shows that the highest accumulation level was obtained for ER-TG2 and vac-TG2, and that cyto-TG2 and sec-TG2 levels were approximately 9- to 16-fold lower than those of the ER and vac variants. No significant differences were observed between ER-TG2 and vac-TG2, suggesting that fusion to either SEKDEL or KISIA C-terminal signals is equally efficient to increase TG2 accumulation levels. A kinetic analysis of ER-TG2 and vac-TG2 expression showed that maximum accumulation was reached at 5 d.p.i (Supplementary Figure S2).


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)

Subcellular targeting strategies tested for stabilize TG2 in leaves. (A) Schematic representation of the TG2 constructs used for Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. Cyto-TG2 is a cytosolic form of TG2. Sec-TG2, ER-TG2, and Vac-TG2 are introduced in the secretory pathway with murine signal peptide (SP) from gamma 1 antibody chain; SEKDEL, ER retention SP; KISIA is a CT vacuolar targeting signal of the amaranth 11S globulin. Scheme is not drawn to scale. (B) Enzyme-linked Immunosorbent Assay (ELISA) of TG2 fused to the different sorting signals. Microwells were coated with the same amount of total leaves extract overnight at 4°C. After blocking, anti-TG2 mAb 2G3 was added, followed of incubation with a biotin-conjugated anti-mouse, later with HRP-conjugated streptavidin and developed with TMB peroxidase substrate. Three biological replicates (each replicate containing five leaf disks of the infiltrated tissue from a different plant) were used for ELISA. Error bars represent the standard error of the mean (SEM). ∗∗∗∗Denotes statistically significant difference by Tukey’s multiple comparisons test (P < 0.001). (C) Western blot of TG2 fused to the different sorting signals. Expression levels were measured by scanning densitometry of Western Blot developed with 2G3 mAb with a minimum of three independent experiments. The amount of total extract was adjusted using RLS stained with Coomassie Brilliant Blue R-250 as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Subcellular targeting strategies tested for stabilize TG2 in leaves. (A) Schematic representation of the TG2 constructs used for Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. Cyto-TG2 is a cytosolic form of TG2. Sec-TG2, ER-TG2, and Vac-TG2 are introduced in the secretory pathway with murine signal peptide (SP) from gamma 1 antibody chain; SEKDEL, ER retention SP; KISIA is a CT vacuolar targeting signal of the amaranth 11S globulin. Scheme is not drawn to scale. (B) Enzyme-linked Immunosorbent Assay (ELISA) of TG2 fused to the different sorting signals. Microwells were coated with the same amount of total leaves extract overnight at 4°C. After blocking, anti-TG2 mAb 2G3 was added, followed of incubation with a biotin-conjugated anti-mouse, later with HRP-conjugated streptavidin and developed with TMB peroxidase substrate. Three biological replicates (each replicate containing five leaf disks of the infiltrated tissue from a different plant) were used for ELISA. Error bars represent the standard error of the mean (SEM). ∗∗∗∗Denotes statistically significant difference by Tukey’s multiple comparisons test (P < 0.001). (C) Western blot of TG2 fused to the different sorting signals. Expression levels were measured by scanning densitometry of Western Blot developed with 2G3 mAb with a minimum of three independent experiments. The amount of total extract was adjusted using RLS stained with Coomassie Brilliant Blue R-250 as loading control.
Mentions: Although TG2 from Caco2 cell line was cloned in E. coli expression vector and different conditions were assayed to produce it, low recovery levels were obtained. For that reason in this work, we attempted to produce TG2 in plant cells. To this end, four versions of TG2 in plant expression binary vector were obtained: cytosolic (cyto-TG2), secretory (sec-TG2), ER-TG2, and vacuolar (vac-TG2), and their schematic representations are shown in Figure 1A. For vacuolar sorting the C terminal KISIA sequence from the amaranth 11S storage globulin was added to TG2 (Petruccelli et al., 2007). To facilitate purification a six histidine tag was also fused to TG2 (Figure 1A). The four TG2 constructs were introduced in A. tumefaciens GV3101 and leaves of N. benthamiana were infiltrated with these agrobacteria. Five days after infiltration, leaves were collected and accumulation of TG2 was measure by ELISA using anti-TG2 mAb 2G3. Figure 1B shows that the highest accumulation level was obtained for ER-TG2 and vac-TG2, and that cyto-TG2 and sec-TG2 levels were approximately 9- to 16-fold lower than those of the ER and vac variants. No significant differences were observed between ER-TG2 and vac-TG2, suggesting that fusion to either SEKDEL or KISIA C-terminal signals is equally efficient to increase TG2 accumulation levels. A kinetic analysis of ER-TG2 and vac-TG2 expression showed that maximum accumulation was reached at 5 d.p.i (Supplementary Figure S2).

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