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Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli

Abstract : Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h. respectively, were isolated from mid-maturation seed cDNA libraries. The T. aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T. durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5% similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment. anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13 391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5'-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP:thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed alpha-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.
Keywords : REDUCTASE
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https://hal.inrae.fr/hal-02692932
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Marie-Françoise Gautier, Valerie Lullien Pellerin, F. de Lamotte-Guery, Anne-Laure Guirao, Philippe Joudrier. Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli. European Journal of Biochemistry, 1998, 252 (2), pp.314-324. ⟨10.1046/j.1432-1327.1998.2520314.x⟩. ⟨hal-02692932⟩

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