TY - JOUR
T1 - Expression of thioredoxin-fusion proteins of α-gliadin, γ-gliadin and Low Molecular weight glutenin, from wheat endosperm and their domains in enterobacteria
AU - Benitez-Cardoza, Claudia G.
AU - Popineau, Yves
AU - Gueguen, Jacques
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
KW - Heterologous expression
KW - Recombinant gliadin and glutenin
KW - Thioredoxin
KW - Wheat prolamins
UR - http://www.scopus.com/inward/record.url?scp=43949115899&partnerID=8YFLogxK
U2 - 10.3844/ajidsp.2007.84.91
DO - 10.3844/ajidsp.2007.84.91
M3 - Artículo
SN - 1553-6203
VL - 3
SP - 84
EP - 91
JO - American Journal of Infectious Diseases
JF - American Journal of Infectious Diseases
IS - 2
ER -