In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (INIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system The IMP fusion protein, with a poly-l-lis-tag (6x His). was obtained by cloning the coding region of INIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia call BL21 (DE3) pLysS After isolation and purification of the fusion protein by affinity chromatography Using Ni-Sepharose resin, the protein was Purified further using ion exchange chromatography with a Hi-trap resource Q column The recombinant IMP was characterized by Western blotting using three distinct antibodies, circular dichioism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning. (C) 2009 Elsevier Inc All rights reserved.