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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

Induction of protein bodies by expression of ELP. Expression of ER-GFP and sec-RFP (A), ER-RFP-TG2 and ER-GFP (B), ER-GFP, sec-RFP and ELP (C), ER-RFP-TG2, ER-GFP and ELP (D) in N. benthamiana leaf epidermal cells. ER-GFP shows the typical ER reticulated pattern (green channel, A), sec-RFP has an irregular pattern on the borders of the cell typical of apoplast (apo; red channel, A), no co-localization of ER-GFP and sec-RFP is observed in the merge panel. ER-RFP-TG2 (B) is mainly located in clusters (PB) in the borders of the cells (arrows), and the signal on the rest of the ER network is low. Co-localization of ER-RFP-TG2 and ER-GFP is observed mainly in these clusters (B, merge). ELP expression induces protein body formation (PB) in (C,D). Co-localization of ER-GFP and sec-RFP is observed in PB (C, merge) while RFP-TG2 did not entirely colocalize with ER-GFP (D, merge) in the nuclear region of the cell. Scale bars: 10 μm (A,B) and 5 μm (C,D).
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Figure 2: Induction of protein bodies by expression of ELP. Expression of ER-GFP and sec-RFP (A), ER-RFP-TG2 and ER-GFP (B), ER-GFP, sec-RFP and ELP (C), ER-RFP-TG2, ER-GFP and ELP (D) in N. benthamiana leaf epidermal cells. ER-GFP shows the typical ER reticulated pattern (green channel, A), sec-RFP has an irregular pattern on the borders of the cell typical of apoplast (apo; red channel, A), no co-localization of ER-GFP and sec-RFP is observed in the merge panel. ER-RFP-TG2 (B) is mainly located in clusters (PB) in the borders of the cells (arrows), and the signal on the rest of the ER network is low. Co-localization of ER-RFP-TG2 and ER-GFP is observed mainly in these clusters (B, merge). ELP expression induces protein body formation (PB) in (C,D). Co-localization of ER-GFP and sec-RFP is observed in PB (C, merge) while RFP-TG2 did not entirely colocalize with ER-GFP (D, merge) in the nuclear region of the cell. Scale bars: 10 μm (A,B) and 5 μm (C,D).

Mentions: A novel ELP construct consisting in 36 repeats of the pentapeptide VPGXG in which the guest residues X were V and F in ratio 8:1 (Supplementary Figure S1) with a theoretical inverse phase transition temperature (Tt) of 18°C (Urry et al., 1992) and which is expected to be insoluble at N. benthamiana growing conditions was used in this work. The ELP was sorted to the ER by means of a secretory SP and SEKDEL ER retention sequence. The ability of this ELP to induce protein body formation was analyzed with CLSM, using GFP-HDEL (Haseloff et al., 1997) and sec-RFP (Scabone et al., 2011) as fluorescent markers of the secretory pathway. Figure 2A shows that ER-GFP had a normal reticular pattern in the absence of ELP and that sec-RFP localized on the borders of the cell with an irregular pattern typical of apoplast accumulation. ER-RFP-TG2 had also a reticular pattern but its accumulation produced clusters on the borders of the cells (Figure 2B, arrows). A partial co-localization was observed between ER-GFP and ER-RFP-TG2 in the merge panel, ER-RFP-TG2 was located mainly in the clusters while ER-GFP had an uniform distribution (Figure 2B). Accumulation of ER-RFP-TG2 fusion was approximately 8,4 ± 1,8 μg/g fresh leaf tissue. When ER-GFP and sec-RFP were co-expressed with ELP, large ER-PB were observed predominantly close to the nuclei and in cortical regions (Figure 2C). A co-localization pattern of sec-RFP in transit with ER-GFP was found as can be observed in yellow in the merge panel (Figure 2C). Nevertheless ELP did not affect final localization of sec-RFP since the apo pattern was also observed for this construct (Supplementary Figure S3). Some of the ELP induced PBs were larger than the nucleolus (Figure 2C). When ER-RFP-TG2 was co-expressed with ELP, small (less than 1 μm) and large PBs were also observed (Figure 2D) but only a partial co-localization with ER-GFP was detected (Figure 2D, merge panel). PBs, in the nuclear region, had heterogeneous size and composition distribution since some of them had only ER-RFP-TG2 and other only ER-GFP. In contrast, in the cortical region, a complete co-localization of green ER-GFP PBs and red ER-RFP-TG2 PBs was observed (Supplementary Figure S4). The integrity of ER-RFP-TG2 was confirmed by immunoblot analysis to ensure that the red fluorescence corresponded to entire fusion protein (Supplementary Figure S5).


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)

Induction of protein bodies by expression of ELP. Expression of ER-GFP and sec-RFP (A), ER-RFP-TG2 and ER-GFP (B), ER-GFP, sec-RFP and ELP (C), ER-RFP-TG2, ER-GFP and ELP (D) in N. benthamiana leaf epidermal cells. ER-GFP shows the typical ER reticulated pattern (green channel, A), sec-RFP has an irregular pattern on the borders of the cell typical of apoplast (apo; red channel, A), no co-localization of ER-GFP and sec-RFP is observed in the merge panel. ER-RFP-TG2 (B) is mainly located in clusters (PB) in the borders of the cells (arrows), and the signal on the rest of the ER network is low. Co-localization of ER-RFP-TG2 and ER-GFP is observed mainly in these clusters (B, merge). ELP expression induces protein body formation (PB) in (C,D). Co-localization of ER-GFP and sec-RFP is observed in PB (C, merge) while RFP-TG2 did not entirely colocalize with ER-GFP (D, merge) in the nuclear region of the cell. Scale bars: 10 μm (A,B) and 5 μm (C,D).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Induction of protein bodies by expression of ELP. Expression of ER-GFP and sec-RFP (A), ER-RFP-TG2 and ER-GFP (B), ER-GFP, sec-RFP and ELP (C), ER-RFP-TG2, ER-GFP and ELP (D) in N. benthamiana leaf epidermal cells. ER-GFP shows the typical ER reticulated pattern (green channel, A), sec-RFP has an irregular pattern on the borders of the cell typical of apoplast (apo; red channel, A), no co-localization of ER-GFP and sec-RFP is observed in the merge panel. ER-RFP-TG2 (B) is mainly located in clusters (PB) in the borders of the cells (arrows), and the signal on the rest of the ER network is low. Co-localization of ER-RFP-TG2 and ER-GFP is observed mainly in these clusters (B, merge). ELP expression induces protein body formation (PB) in (C,D). Co-localization of ER-GFP and sec-RFP is observed in PB (C, merge) while RFP-TG2 did not entirely colocalize with ER-GFP (D, merge) in the nuclear region of the cell. Scale bars: 10 μm (A,B) and 5 μm (C,D).
Mentions: A novel ELP construct consisting in 36 repeats of the pentapeptide VPGXG in which the guest residues X were V and F in ratio 8:1 (Supplementary Figure S1) with a theoretical inverse phase transition temperature (Tt) of 18°C (Urry et al., 1992) and which is expected to be insoluble at N. benthamiana growing conditions was used in this work. The ELP was sorted to the ER by means of a secretory SP and SEKDEL ER retention sequence. The ability of this ELP to induce protein body formation was analyzed with CLSM, using GFP-HDEL (Haseloff et al., 1997) and sec-RFP (Scabone et al., 2011) as fluorescent markers of the secretory pathway. Figure 2A shows that ER-GFP had a normal reticular pattern in the absence of ELP and that sec-RFP localized on the borders of the cell with an irregular pattern typical of apoplast accumulation. ER-RFP-TG2 had also a reticular pattern but its accumulation produced clusters on the borders of the cells (Figure 2B, arrows). A partial co-localization was observed between ER-GFP and ER-RFP-TG2 in the merge panel, ER-RFP-TG2 was located mainly in the clusters while ER-GFP had an uniform distribution (Figure 2B). Accumulation of ER-RFP-TG2 fusion was approximately 8,4 ± 1,8 μg/g fresh leaf tissue. When ER-GFP and sec-RFP were co-expressed with ELP, large ER-PB were observed predominantly close to the nuclei and in cortical regions (Figure 2C). A co-localization pattern of sec-RFP in transit with ER-GFP was found as can be observed in yellow in the merge panel (Figure 2C). Nevertheless ELP did not affect final localization of sec-RFP since the apo pattern was also observed for this construct (Supplementary Figure S3). Some of the ELP induced PBs were larger than the nucleolus (Figure 2C). When ER-RFP-TG2 was co-expressed with ELP, small (less than 1 μm) and large PBs were also observed (Figure 2D) but only a partial co-localization with ER-GFP was detected (Figure 2D, merge panel). PBs, in the nuclear region, had heterogeneous size and composition distribution since some of them had only ER-RFP-TG2 and other only ER-GFP. In contrast, in the cortical region, a complete co-localization of green ER-GFP PBs and red ER-RFP-TG2 PBs was observed (Supplementary Figure S4). The integrity of ER-RFP-TG2 was confirmed by immunoblot analysis to ensure that the red fluorescence corresponded to entire fusion protein (Supplementary Figure S5).

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