Mitochondrial intermediate peptidase: expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates.
In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli 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 hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, 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.
Marcondes
Marcelo F
MF
Department of Biophysics, Federal University of São Paulo, São Paulo 04044-020, Brazil.
Torquato
Ricardo J S
RJ
Assis
Diego M
DM
Juliano
Maria A
MA
Hayashi
Mirian A F
MA
Oliveira
Vitor
V
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C068038:mitochondrial intermediate peptidase