Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli

Sambanthamurthy Soundar, Gary T. Jennings, Lee McAlister-Henn, Roberta F. Colman

Research output: Contribution to journalArticle

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Abstract

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 enzyme 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 dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25°C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-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 procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a K(m) for DL-isocitrate of 7.74 ± 0.18 μM and a K(m) for NADP+ of 6.63 ± 1.34 μM. These values are similar to the K(m)s measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

Original languageEnglish (US)
Pages (from-to)305-312
Number of pages8
JournalProtein Expression and Purification
Volume8
Issue number3
DOIs
StatePublished - Nov 1996

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ASJC Scopus subject areas

  • Biotechnology

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