Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence.
Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells. Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.
Rouhier
Nicolas
N
Unité Mixte de Recherche 1136 IaM (Interaction Arbres Microorganismes), INRA-UHP Nancy I. Université Henri Poincaré, Vandoeuvre, France.
Gelhaye
Eric
E
Sautière
Pierre-Eric
PE
Jacquot
Jean-Pierre
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli; D054477:Glutaredoxins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D017422:Sequence Analysis, DNA; D014197:Trees
D018076:DNA, Complementary; D054477:Glutaredoxins; D011506:Proteins; D010088:Oxidoreductases