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

• Published in: European Journal of Biochemistry 1998-03, Vol.252 (2), p.314-324
• Main Authors: Gautier, Marie‐Françoise, Lullien‐Pellerin, Valérie, de Lamotte‐Guéry, Frédéric, Guirao, Anne, Joudrier, Philippe
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biochemistry, Molecular Biology; Bridged Bicyclo Compounds - metabolism; cDNA sequence; Cloning, Molecular; disulfide reduction; Disulfides - metabolism; Dithionitrobenzoic Acid - metabolism; Escherichia coli; Escherichia coli - genetics; Fluorescent Dyes - metabolism; Kinetics; Life Sciences; Molecular Sequence Data; Mutagenesis, Site-Directed; Plant Proteins - chemistry; plasmid pTaM13.38; plasmid pTd14.13.2; recombinant protein; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Analysis, DNA; thioredoxin h; Thioredoxin-Disulfide Reductase - metabolism; Thioredoxins - chemistry; Triticum - chemistry; Triticum aestivum; Triticum durum; wheat
• ISSN: 0014-2956; 1432-1033; 1432-1327
• DOI: 10.1046/j.1432-1327.1998.2520314.x

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 α‐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.