Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli.
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.
Gautier
M F
MF
Unité de Biochimie et Biologie Moléculaire des Céréales, INRA, Montpellier, France. gautier@ensam.inra.fr
Lullien-Pellerin
V
V
de Lamotte-Guéry
F
F
Guirao
A
A
Joudrier
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry*; D014908:Triticum / Q000737:chemistry*
D001643:Bridged Bicyclo Compounds; D004220:Disulfides; D005456:Fluorescent Dyes; D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; C028381:monobromobimane