title: Purification and characterization of a fully active recombinant tobacco cytosolic NADP-dependent isocitrate dehydrogenase in Escherichia coli: evidence for a role for the N-terminal region in enzyme activity.
abstract: The recently isolated full-length NADP-dependent isocitrate dehydrogenase (ICDH) cDNA encoding the tobacco cytosolic isoenzyme has been cloned into the expression vector pET8c and used to transform Escherichia coli strain BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and used to raise polyclonal antibodies. Its kinetic properties were found to be identical to those of the cytosolic ICDH isoenzyme purified from tobacco cell cultures. The recombinant and the endogenous bacterial ICDH could be easily distinguished by their different behaviors during anion-exchange column chromatography and immunological response. An incomplete ICDH-encoding cDNA clone, encoding a protein lacking the first 36 amino acids at the N-terminus, was cloned into the expression vector pKK233-2 and used to transform ICDH-lacking E. coli cells (strain 2004). The truncated, recombinant ICDH produced by the bacteria was found to be inactive.
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase