Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

Author Marcondes, Marcelo F. Autor UNIFESP Google Scholar
Torquato, Ricardo J. S. Autor UNIFESP Google Scholar
Assis, Diego M. Autor UNIFESP Google Scholar
Juliano, Maria A. Autor UNIFESP Google Scholar
Hayashi, Mirian A. F. Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract 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.
Keywords Peptidase
Mitochondria
Bacterial expression
Enzymatic activity
FRET substrate
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Date 2010-01-01
Published in Biochemical and Biophysical Research Communications. San Diego: Academic Press Inc Elsevier Science, v. 391, n. 1, p. 123-128, 2010.
ISSN 0006-291X (Sherpa/Romeo, impact factor)
Publisher Elsevier B.V.
Extent 123-128
Origin http://dx.doi.org/10.1016/j.bbrc.2009.11.014
Access rights Closed access
Type Article
Web of Science ID WOS:000273624500023
URI http://repositorio.unifesp.br/handle/11600/32086

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