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Expression of a new chimeric protein with a highly repeated sequence in tobacco cells

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Abstract

In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GSW) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GSM-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

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Abbreviations

BiP:

Binding protein

BY-2:

Bright yellow-2

ELP:

Elastin-like peptide

ER:

Endoplasmic reticulum

GFP:

Green fluorescent protein

GSW :

Wild-type glutenin subunit

GSM :

Modified glutenin subunit

GSM-ELP:

Modified glutenin subunit-elastin like peptide

HMW:

High-molecular weight

PB:

Protein body

PDI:

Protein disulphide isomerase

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Acknowledgments

The authors thank Denis Lourdin for the discussion of the triblock protein design and properties, and Peter Shewry and Paola Tosi for the critical review of the manuscript. We also thank Axelle Bouder for her technical assistance in molecular and cellular biology experiments, Brigitte Bouchet and Marie-Françoise Devaux for their assistance with confocal microscopy and fluorescence quantification, respectively. We gratefully acknowledge MESR for the post-doctoral grant of A. Saumonneau.

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Authors and Affiliations

  1. INRA, UR1268, Biopolymères Interactions Assemblages, 44300, Nantes, France

    Amélie Saumonneau, Karine Rottier, Yves Popineau, Jacques Guéguen & Mathilde Francin-Allami

  2. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466, Gatersleben, Germany

    Udo Conrad

Authors
  1. Amélie Saumonneau
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  2. Karine Rottier
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  3. Udo Conrad
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  4. Yves Popineau
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  5. Jacques Guéguen
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  6. Mathilde Francin-Allami
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Correspondence to Jacques Guéguen or Mathilde Francin-Allami.

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Communicated by F. Brandizzi.

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Saumonneau, A., Rottier, K., Conrad, U. et al. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells. Plant Cell Rep 30, 1289–1302 (2011). https://doi.org/10.1007/s00299-011-1040-z

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  • DOI: https://doi.org/10.1007/s00299-011-1040-z

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