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A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway.

Moeller L, Gan Q, Wang K - J. Exp. Bot. (2009)

Bottom Line: Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions.This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway.The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

View Article: PubMed Central - PubMed

Affiliation: Iowa State University, Ames, IA 50011-1010, USA.

ABSTRACT
The Escherichia coli heat-labile enterotoxin B subunit (LT-B) has been used as a model antigen for the production of plant-derived high-valued proteins in maize. LT-B with its native signal peptide (BSP) has been shown to accumulate in starch granules of transgenic maize kernels. To elucidate the targeting properties of the bacterial LT-B protein and BSP in plant systems, the subcellular localization of visual marker green fluorescent protein (GFP) fused to LT-B and various combinations of signal peptides was examined in Arabidopsis protoplasts and transgenic maize. Biochemical analysis indicates that the LT-B::GFP fusion proteins can assemble and fold properly retaining both the antigenicity of LT-B and the fluorescing properties of GFP. Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions. Confocal microscopy analysis indicates that the fusion proteins accumulate in the endomembrane system of plant cells in a signal peptide-dependent fashion. This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway. The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

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Related in: MedlinePlus

Gene expression analyses of LT-B, GFP, and LT-B::GFP fusions in transgenic maize kernels. (A) Bright field and fluorescence imaging of a representative self-pollinated ear of transgenic line P310 (Pγzein-BSP-LT-B::GFP) expressing GFP in the endosperm. A GFP-expressing kernel is marked by a white arrow. (B) LT-B levels as a percentage of total aqueous extractable protein (% LT-B/TAEP) in endosperm of P310 and P311 (Pγzein-ZSP-LT-B::GFP) kernels. Both transgenic maize carrying BSP- or ZSP-led LT-B::GFP fusion protein show the expression of functional LT-B. However, independent lines from both constructs have different levels of LT-B. (C) Western blot of TAEP extracts from transgenic callus (P308c, P35S-BSP-LT-B::GFP) and endosperms (P309, Pγzein-GFP, GFP control; P310-28, P310-32, two independent lines from P310; P311; and P315, Pγzein-BSP-GFP) using anti-GFP antibody. (D) Western blot of TAEP extracts from transgenic callus (P308c) and endosperms (P309, P310-32, and P315) using anti-LT-B antibody. (E) Western blot of immuno-precipitated samples using anti-LT-B antibody, probed with anti-GFP antibody. B73, non-transgenic maize line. P77, transgenic maize line expressing LT-B with its native bacterial signal peptide. The empty lane in (D) was a sample lost during loading. LT-B std, bacterial LT-B protein standard. EGFP std, commercial enhanced GFP standard. Arrowheads in (C), GFP. Dots in (C), possible cleavage peptides cross-react to GFP antibody. Asterisks in (C), (D), and (E), LT-B::GFP fusion. Open diamonds in (D), LT-B monomer. Closed diamond in (D), truncated LT-B::GFP fusion. Open circle in (D), LT-B multimer. Arrow in (E), commercial EGFP. Multiple EGFP bands in GFP standard may due to incomplete protein denaturation during boiling before loading.
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fig2: Gene expression analyses of LT-B, GFP, and LT-B::GFP fusions in transgenic maize kernels. (A) Bright field and fluorescence imaging of a representative self-pollinated ear of transgenic line P310 (Pγzein-BSP-LT-B::GFP) expressing GFP in the endosperm. A GFP-expressing kernel is marked by a white arrow. (B) LT-B levels as a percentage of total aqueous extractable protein (% LT-B/TAEP) in endosperm of P310 and P311 (Pγzein-ZSP-LT-B::GFP) kernels. Both transgenic maize carrying BSP- or ZSP-led LT-B::GFP fusion protein show the expression of functional LT-B. However, independent lines from both constructs have different levels of LT-B. (C) Western blot of TAEP extracts from transgenic callus (P308c, P35S-BSP-LT-B::GFP) and endosperms (P309, Pγzein-GFP, GFP control; P310-28, P310-32, two independent lines from P310; P311; and P315, Pγzein-BSP-GFP) using anti-GFP antibody. (D) Western blot of TAEP extracts from transgenic callus (P308c) and endosperms (P309, P310-32, and P315) using anti-LT-B antibody. (E) Western blot of immuno-precipitated samples using anti-LT-B antibody, probed with anti-GFP antibody. B73, non-transgenic maize line. P77, transgenic maize line expressing LT-B with its native bacterial signal peptide. The empty lane in (D) was a sample lost during loading. LT-B std, bacterial LT-B protein standard. EGFP std, commercial enhanced GFP standard. Arrowheads in (C), GFP. Dots in (C), possible cleavage peptides cross-react to GFP antibody. Asterisks in (C), (D), and (E), LT-B::GFP fusion. Open diamonds in (D), LT-B monomer. Closed diamond in (D), truncated LT-B::GFP fusion. Open circle in (D), LT-B multimer. Arrow in (E), commercial EGFP. Multiple EGFP bands in GFP standard may due to incomplete protein denaturation during boiling before loading.

Mentions: To examine whether LT-B and GFP retain their functional properties in the fusions described, experiments were carried out to assess their correct folding and assembly in maize. In stably transformed callus, green fluorescence could easily be distinguished when the fusion constructs were present (data not shown). Similarly, it was possible to detect green fluorescent transgenic maize seeds (Fig. 2A) confirming that GFP, a molecule capable of folding and fluorescing without exogenous substrates or cofactors (Chalfie et al., 1994) is active and correctly folded.


A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway.

Moeller L, Gan Q, Wang K - J. Exp. Bot. (2009)

Gene expression analyses of LT-B, GFP, and LT-B::GFP fusions in transgenic maize kernels. (A) Bright field and fluorescence imaging of a representative self-pollinated ear of transgenic line P310 (Pγzein-BSP-LT-B::GFP) expressing GFP in the endosperm. A GFP-expressing kernel is marked by a white arrow. (B) LT-B levels as a percentage of total aqueous extractable protein (% LT-B/TAEP) in endosperm of P310 and P311 (Pγzein-ZSP-LT-B::GFP) kernels. Both transgenic maize carrying BSP- or ZSP-led LT-B::GFP fusion protein show the expression of functional LT-B. However, independent lines from both constructs have different levels of LT-B. (C) Western blot of TAEP extracts from transgenic callus (P308c, P35S-BSP-LT-B::GFP) and endosperms (P309, Pγzein-GFP, GFP control; P310-28, P310-32, two independent lines from P310; P311; and P315, Pγzein-BSP-GFP) using anti-GFP antibody. (D) Western blot of TAEP extracts from transgenic callus (P308c) and endosperms (P309, P310-32, and P315) using anti-LT-B antibody. (E) Western blot of immuno-precipitated samples using anti-LT-B antibody, probed with anti-GFP antibody. B73, non-transgenic maize line. P77, transgenic maize line expressing LT-B with its native bacterial signal peptide. The empty lane in (D) was a sample lost during loading. LT-B std, bacterial LT-B protein standard. EGFP std, commercial enhanced GFP standard. Arrowheads in (C), GFP. Dots in (C), possible cleavage peptides cross-react to GFP antibody. Asterisks in (C), (D), and (E), LT-B::GFP fusion. Open diamonds in (D), LT-B monomer. Closed diamond in (D), truncated LT-B::GFP fusion. Open circle in (D), LT-B multimer. Arrow in (E), commercial EGFP. Multiple EGFP bands in GFP standard may due to incomplete protein denaturation during boiling before loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2724687&req=5

fig2: Gene expression analyses of LT-B, GFP, and LT-B::GFP fusions in transgenic maize kernels. (A) Bright field and fluorescence imaging of a representative self-pollinated ear of transgenic line P310 (Pγzein-BSP-LT-B::GFP) expressing GFP in the endosperm. A GFP-expressing kernel is marked by a white arrow. (B) LT-B levels as a percentage of total aqueous extractable protein (% LT-B/TAEP) in endosperm of P310 and P311 (Pγzein-ZSP-LT-B::GFP) kernels. Both transgenic maize carrying BSP- or ZSP-led LT-B::GFP fusion protein show the expression of functional LT-B. However, independent lines from both constructs have different levels of LT-B. (C) Western blot of TAEP extracts from transgenic callus (P308c, P35S-BSP-LT-B::GFP) and endosperms (P309, Pγzein-GFP, GFP control; P310-28, P310-32, two independent lines from P310; P311; and P315, Pγzein-BSP-GFP) using anti-GFP antibody. (D) Western blot of TAEP extracts from transgenic callus (P308c) and endosperms (P309, P310-32, and P315) using anti-LT-B antibody. (E) Western blot of immuno-precipitated samples using anti-LT-B antibody, probed with anti-GFP antibody. B73, non-transgenic maize line. P77, transgenic maize line expressing LT-B with its native bacterial signal peptide. The empty lane in (D) was a sample lost during loading. LT-B std, bacterial LT-B protein standard. EGFP std, commercial enhanced GFP standard. Arrowheads in (C), GFP. Dots in (C), possible cleavage peptides cross-react to GFP antibody. Asterisks in (C), (D), and (E), LT-B::GFP fusion. Open diamonds in (D), LT-B monomer. Closed diamond in (D), truncated LT-B::GFP fusion. Open circle in (D), LT-B multimer. Arrow in (E), commercial EGFP. Multiple EGFP bands in GFP standard may due to incomplete protein denaturation during boiling before loading.
Mentions: To examine whether LT-B and GFP retain their functional properties in the fusions described, experiments were carried out to assess their correct folding and assembly in maize. In stably transformed callus, green fluorescence could easily be distinguished when the fusion constructs were present (data not shown). Similarly, it was possible to detect green fluorescent transgenic maize seeds (Fig. 2A) confirming that GFP, a molecule capable of folding and fluorescing without exogenous substrates or cofactors (Chalfie et al., 1994) is active and correctly folded.

Bottom Line: Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions.This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway.The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

View Article: PubMed Central - PubMed

Affiliation: Iowa State University, Ames, IA 50011-1010, USA.

ABSTRACT
The Escherichia coli heat-labile enterotoxin B subunit (LT-B) has been used as a model antigen for the production of plant-derived high-valued proteins in maize. LT-B with its native signal peptide (BSP) has been shown to accumulate in starch granules of transgenic maize kernels. To elucidate the targeting properties of the bacterial LT-B protein and BSP in plant systems, the subcellular localization of visual marker green fluorescent protein (GFP) fused to LT-B and various combinations of signal peptides was examined in Arabidopsis protoplasts and transgenic maize. Biochemical analysis indicates that the LT-B::GFP fusion proteins can assemble and fold properly retaining both the antigenicity of LT-B and the fluorescing properties of GFP. Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions. Confocal microscopy analysis indicates that the fusion proteins accumulate in the endomembrane system of plant cells in a signal peptide-dependent fashion. This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway. The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

Show MeSH
Related in: MedlinePlus