Expression of a cDNA encoding the wheat CM16 protein in Escherichia coli.
The wheat kernel CM16 protein, a subunit of the heterotetrameric insect alpha-amylase inhibitor that has been involved in the technological quality of wheat-products, was produced in Escherichia coli. Cloning of the cDNA part encoding the mature protein in a pET expression plasmid, under the control of a promoter for the bacteriophage T7 RNA polymerase, allows the synthesis of large amounts of the CM16 protein in the bacteria. Upon induction with isopropyl thiogalactopyranoside the recombinant protein accumulates in insoluble inclusion bodies. Solubilization with 6 M urea containing 0.5 mM dithiothreitol, followed by slow elimination of the denaturing agents by step dialysis, results in a significant recovery of the recombinant protein in a soluble, monomeric form. Characterization of the protein was done by automated Edman degradation and total amino acid determination. The recombinant protein in comparison with the one isolated from wheat exhibits a Met extension at the N-terminus that was introduced in the construction for translation initiation. The CM16 protein produced in this manner has the advantage over wheat purified protein of not being contaminated with other proteins from the same family and constitutes adequate material for further analysis of the technological properties of the protein in wheat-derived products.
Lullien-Pellerin
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V
Laboratoire de Technologie des Céréales, INRA, Montpellier, France.
Gavalda
S
S
Joudrier
P
P
Gautier
M F
MF
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012639:Seeds / Q000378:metabolism; D014908:Triticum / Q000235:genetics / Q000378:metabolism*; D014508:Urea
D000596:Amino Acids; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D010940:Plant Proteins; D011994:Recombinant Proteins; D014508:Urea; D004229:Dithiothreitol