Expression of Thioredoxin-Fusion Proteins of α-Gliadin, γ-Gliadin and Low Molecular Weight Glutenin, from Wheat Endosperm and their Domains in Enterobacteria
- 1 Instituto Politécnico Nacional, Mexico
- 2 Unité Biopolymères Interactions Assemblages, Mexico
Abstract
Wheat seed storage proteins play a determining role in the viscoelastic properties of wheat gluten. The genes encoding α-gliadin, γ-gliadin and Low Molecular Weight glutenin and their N-central-repetitive and C-terminal domains from wheat endosperm had been subcloned into a thioredoxin expression system (pET102/D-Topo) and produced as fusion proteins in E. coli. The expression levels for each of the proteins varied among constructs from 5 to 12 % of the total proteins in E. coli. This indicates that obtaining prolamins as fusion proteins to thioredoxin might have the potential for preparing milligram quantities of the proteins tested here. The identity of the synthesized polypeptides was confirmed by immunoblotting and antibody-cross reactions. Two cleavage methods for the removal of thioredoxin were assayed. Nevertheless, the attempts to remove the fusion partner from most of the constructs failed. The only construct that was able to be cleaved either by Entorokinase, or by acid cleavage, was the N-terminal domain of γ-Gliandin. Also this construct showed enhanced solubility compared with the rest of the polypeptides produced. Some aspects of the sequence that might contribute to the different behaviour of this construct are discussed. The results presented in this work open new alternatives for the production of large amounts of seed storage proteins, in order to further characterise their structure and interactions.
DOI: https://doi.org/10.3844/ajidsp.2007.84.91
Copyright: © 2007 Claudia G. Benitez-Cardoza, Yves Popineau and Jacques Gueguen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Keywords
- Wheat prolamins
- heterologous expression
- thioredoxin
- recombinant gliadin and glutenin