Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of γ-Gliadin

Benítez‐Cardoza, Claudia G.; Rogniaux, Hélène; Popineau, Yves; Guéguen, Jacques

doi:10.1016/j.pep.2005.08.017

Cited by 6 publications

(4 citation statements)

References 52 publications

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“…This indicates a major conformation change caused mainly by the solvent, since the reducing agent was used in all solutions. The spectrum of γ‐zein in water is not so similar to the ones observed to proteins with PPII conformation 5, 6, 24. It is consistent with a protein containing approximately 50% α‐helix and unordered PPII conformation.…”

Section: Resultsmentioning

confidence: 54%

γ‐Zein secondary structure in solution by circular dichroism

Bicudo

Forato

et al. 2007

Biopolymers

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The proline-rich N-terminal domain of gamma-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution.

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Section: Resultsmentioning

confidence: 54%

γ‐Zein secondary structure in solution by circular dichroism

Bicudo

Forato

et al. 2007

Biopolymers

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“…This indicates that EK cleavage might have not worked due to inaccessibility of the protease to its specific cleavage site. [20] In order to sort this problem out, an alternative would be to assay further experimental conditions for EK cleavage, such as pH, buffer composition and temperature. Another alternative could be to replace the Enterokinase site of the constructs by the specific cleavage site of another enzyme using molecular biology techniques.…”

Section: Resultsmentioning

confidence: 99%

“…The results obtained with this construct are thoroughly discussed elsewhere. [20] DISCUSSION Seed storage proteins are considered the most important determinants of the viscoelastic properties of wheat gluten and, as such, of bread-making quality. Glutenins and gliadins interact to form a protein network into the ripe grain, precursor of the mentioned rheological features of gluten and wheat flour.…”

Section: Resultsmentioning

confidence: 99%

Expression of Thioredoxin-Fusion Proteins of α-Gliadin, γ-Gliadin and Low Molecular Weight Glutenin, from Wheat Endosperm and their Domains in Enterobacteria

Benítez‐Cardoza¹,

Popineau²,

Guéguen³

2007

American J. of Infectious Diseases

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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 Ncentral-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.

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“…On the other hand, the promoter, only expressed in a very low level of polypeptides [37]. However, being fused with thioredoxin gene without low compositional complexity segment, spr and the nucleotide sequence encoding the N-terminal repetitive domain of γ-gliadin could be over-expressed as fusion proteins in E. coli [38,39]. It was also reported that using the pelB, ompA or CSP signal sequence as the leading sequence, both insoluble and soluble proteins of GM-CSF and scFv-phOx were achieved at high level, but no product was detected when the signal peptide was absent [40].…”

Section: Discussionmentioning

confidence: 99%

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5′ end in Escherichia coli

Wang

Gao

et al. 2015

Protein Expression and Purification

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractWheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in E. coli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 2 9 genes, viz. 6 LMW-GS and 3 α-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5'end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP*-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.

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Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of γ-Gliadin

Cited by 6 publications

References 52 publications

γ‐Zein secondary structure in solution by circular dichroism

γ‐Zein secondary structure in solution by circular dichroism

Expression of Thioredoxin-Fusion Proteins of α-Gliadin, γ-Gliadin and Low Molecular Weight Glutenin, from Wheat Endosperm and their Domains in Enterobacteria

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5′ end in Escherichia coli

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