EXPRESSION OF PIG-HEART MITOCHONDRIAL NADP-DEPENDENT ISOCITRATE DEHYDROGENASE IN ESCHERICHIA-COLI

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases . The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific e nzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor X (a) between the maltose binding protein and isocitrate dehydrogenase; however, since Factor X(a) was also found to digest isocitrate dehydro genase, a thrombin recognition site was substituted. The fusion protei n was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenas e by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion prote in with thrombin generated pig heart NADP-specific isocitrate dehydrog enase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A il-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification proc edure. This enzyme exhibits a single band (M(r) = 46,600) on polyacryl amide gel electrophoresis in the presence of sodium dodecyl sulfate an d, under standard assay conditions, has a K-m for DL-isocitrate of 7.7 4 +/- 0.18 mu M and a K-m for NADP(+) of 6.63 +/- 1.34 mu M. These val ues are similar to the K(m)s measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identic al with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that t his expression and purification system yields pure pig heart mitochond rial NADP-specific isocitrate dehydrogenase and should allow generatio n of wild-type and mutant enzymes in amounts suitable for their bioche mical characterization and comparison. (C) 1996 Academic Press, Inc.