Properties of recombinant NADP-malate dehydrogenases from Sorghum vulgare leaves expressed in Escherichia coli cells.
In this study, a cDNA clone coding for sorghum leaf NADP-malate dehydrogenase [Crétin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallantin, M., Huet, J. C. & Pernollet, J. C. (1990) Eur. J. Biochem. 192, 299-303] was used either in the full-length form or in a shorter form deprived of the 5' end coding for the transit peptide. Both cDNA fragments were cloned into the expression vector pKK233-2 and the resulting constructions were used to transform E. coli cells. The bacterial cells which do not contain any NADP-dependent malate dehydrogenase before transformation were able to express the protein after transformation and induction, as detected both by activity measurements and by immunoblot. The recombinant proteins could be purified to homogeneity and their biochemical characteristics studied. They were identical to those of the enzyme isolated from corn or sorghum leaves, including the well known redox regulatory properties. The NADP-malate dehydrogenases derived from both constructions had a similar subunit size and the analysis of their N-terminal sequences revealed that E. coli cells were able to recognize the processing signal of the precursor polypeptide and to mature and assemble the protein in a manner similar to higher plants.
Jacquot
J P
JP
Physiologie Végétale Moléculaire UA CNRS 1128, Orsay, France.
Keryer
E
E
Issakidis
E
E
Decottignies
P
P
Miginiac-Maslow
M
M
Schmitter
J M
JM
Crétin
C
C
D000595:Amino Acid Sequence; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008291:Malate Dehydrogenase / Q000235:genetics*; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D004247:DNA; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)