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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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Gene cassettes, plasmids, and transgenic lines for studying localization of LT-B using GFP as a reporter. Gene cassettes A–E were used in transient assays using Arabidopsis leaf and root protoplasts, and in stable transformation of maize callus and endosperm tissues. Constitutive expression in Arabidopsis protoplasts and in maize callus was driven by the double CaMV 35S promoter (P35S promoter). The maize 27 kDa γ-zein promoter (Pγzein promoter) was used to drive expression of gene cassettes A–E in maize endosperm. The number of independent transgenic events for each line is presented in parenthesis. TEV, tobacco etch virus translational enhancer leader sequence; EGFP, enhanced green fluorescent protein; Tvsp, the soybean vegetative storage protein terminator; BSP, the LT-B bacterial signal peptide; LT-B, the B subunit of E. coli heat-labile enterotoxin; ZSP, 27 kDa γ-zein signal peptide; AG linker, alanine-glycine linker; na, not available.
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fig1: Gene cassettes, plasmids, and transgenic lines for studying localization of LT-B using GFP as a reporter. Gene cassettes A–E were used in transient assays using Arabidopsis leaf and root protoplasts, and in stable transformation of maize callus and endosperm tissues. Constitutive expression in Arabidopsis protoplasts and in maize callus was driven by the double CaMV 35S promoter (P35S promoter). The maize 27 kDa γ-zein promoter (Pγzein promoter) was used to drive expression of gene cassettes A–E in maize endosperm. The number of independent transgenic events for each line is presented in parenthesis. TEV, tobacco etch virus translational enhancer leader sequence; EGFP, enhanced green fluorescent protein; Tvsp, the soybean vegetative storage protein terminator; BSP, the LT-B bacterial signal peptide; LT-B, the B subunit of E. coli heat-labile enterotoxin; ZSP, 27 kDa γ-zein signal peptide; AG linker, alanine-glycine linker; na, not available.

Mentions: A schematic representation of the constructs used in this study is presented in Fig. 1. The enhanced green fluorescence protein (EGFP) sequence in pLM01, pLM02, pLM03, pLM08, and pLM09 was cloned from p27zn-signal (Shepherd and Scott, 2009) using standard molecular biology techniques for restriction enzyme-based cloning. Construct pTH210 containing the CaMV 35S promoter (P35S), tobacco etch virus translational enhancer (TEV) and LT-B was used as a cloning vector (Mason et al., 1998) for some of the constructs. For the generation of plasmids pLM01, pLM02, and pLM03 the EGFP sequence was cloned into pTH210 at the NcoI–SacI sites, SacI–SacI sites, and KpnI–SacI sites, respectively. Plasmid pLM01 was used as a backbone for the generation of pLM08 and pLM09. Plasmid pRC5, a pUC19-based vector carrying the maize 27 kDa γ-zein promoter (Pγzein), TEV, the maize 27 kDa γ-zein signal peptide (ZSP) fused to LT-B and the soybean vegetative storage protein terminator (Tvsp), was used as a source of LT-B fused to the maize 27 kDa γ-zein signal peptide (R Chikwamba, unpublished results). The ZSP-LT-B fragment was amplified by the polymerase chain reaction (PCR) method and the digested product was cloned into the NcoI–BstXI site of pLM01. An NcoI–EcoRI fragment of pLM08 was inserted into the NcoI–EcoRI backbone of pLM01 to generate pLM09. A simple alanine-glycine linker (AG linker) consisting of six amino acids with three AG repeats (AGAGAG) was added between LT-B and GFP using oligonucleotide extensions in the PCR primers in plasmids pLM03, pLM08, and pLM09. Expression cassettes (Fig. 1) were cloned and recombined into a Gateway version of pTF101.1 (Paz et al., 2004), pTF101.1gw1, for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plasmid pRC4 (Chikwamba et al., 2002b) was used as a donor of the Pγzein, and derived plasmids.


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 cassettes, plasmids, and transgenic lines for studying localization of LT-B using GFP as a reporter. Gene cassettes A–E were used in transient assays using Arabidopsis leaf and root protoplasts, and in stable transformation of maize callus and endosperm tissues. Constitutive expression in Arabidopsis protoplasts and in maize callus was driven by the double CaMV 35S promoter (P35S promoter). The maize 27 kDa γ-zein promoter (Pγzein promoter) was used to drive expression of gene cassettes A–E in maize endosperm. The number of independent transgenic events for each line is presented in parenthesis. TEV, tobacco etch virus translational enhancer leader sequence; EGFP, enhanced green fluorescent protein; Tvsp, the soybean vegetative storage protein terminator; BSP, the LT-B bacterial signal peptide; LT-B, the B subunit of E. coli heat-labile enterotoxin; ZSP, 27 kDa γ-zein signal peptide; AG linker, alanine-glycine linker; na, not available.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Gene cassettes, plasmids, and transgenic lines for studying localization of LT-B using GFP as a reporter. Gene cassettes A–E were used in transient assays using Arabidopsis leaf and root protoplasts, and in stable transformation of maize callus and endosperm tissues. Constitutive expression in Arabidopsis protoplasts and in maize callus was driven by the double CaMV 35S promoter (P35S promoter). The maize 27 kDa γ-zein promoter (Pγzein promoter) was used to drive expression of gene cassettes A–E in maize endosperm. The number of independent transgenic events for each line is presented in parenthesis. TEV, tobacco etch virus translational enhancer leader sequence; EGFP, enhanced green fluorescent protein; Tvsp, the soybean vegetative storage protein terminator; BSP, the LT-B bacterial signal peptide; LT-B, the B subunit of E. coli heat-labile enterotoxin; ZSP, 27 kDa γ-zein signal peptide; AG linker, alanine-glycine linker; na, not available.
Mentions: A schematic representation of the constructs used in this study is presented in Fig. 1. The enhanced green fluorescence protein (EGFP) sequence in pLM01, pLM02, pLM03, pLM08, and pLM09 was cloned from p27zn-signal (Shepherd and Scott, 2009) using standard molecular biology techniques for restriction enzyme-based cloning. Construct pTH210 containing the CaMV 35S promoter (P35S), tobacco etch virus translational enhancer (TEV) and LT-B was used as a cloning vector (Mason et al., 1998) for some of the constructs. For the generation of plasmids pLM01, pLM02, and pLM03 the EGFP sequence was cloned into pTH210 at the NcoI–SacI sites, SacI–SacI sites, and KpnI–SacI sites, respectively. Plasmid pLM01 was used as a backbone for the generation of pLM08 and pLM09. Plasmid pRC5, a pUC19-based vector carrying the maize 27 kDa γ-zein promoter (Pγzein), TEV, the maize 27 kDa γ-zein signal peptide (ZSP) fused to LT-B and the soybean vegetative storage protein terminator (Tvsp), was used as a source of LT-B fused to the maize 27 kDa γ-zein signal peptide (R Chikwamba, unpublished results). The ZSP-LT-B fragment was amplified by the polymerase chain reaction (PCR) method and the digested product was cloned into the NcoI–BstXI site of pLM01. An NcoI–EcoRI fragment of pLM08 was inserted into the NcoI–EcoRI backbone of pLM01 to generate pLM09. A simple alanine-glycine linker (AG linker) consisting of six amino acids with three AG repeats (AGAGAG) was added between LT-B and GFP using oligonucleotide extensions in the PCR primers in plasmids pLM03, pLM08, and pLM09. Expression cassettes (Fig. 1) were cloned and recombined into a Gateway version of pTF101.1 (Paz et al., 2004), pTF101.1gw1, for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plasmid pRC4 (Chikwamba et al., 2002b) was used as a donor of the Pγzein, and derived plasmids.

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