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

Abstract

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.

Authors:
 [1];  [2]; ;  [1];  [3];  [1]
  1. Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)
  2. Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)
  3. Department of Pharmacology, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)
Publication Date:
OSTI Identifier:
22199955
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 391; Journal Issue: 1; Other Information: Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; CARBOXYLIC ACIDS; CLONING; DICHROISM; ENERGY TRANSFER; ESCHERICHIA COLI; FLUORESCENCE; ION EXCHANGE CHROMATOGRAPHY; MITOCHONDRIA; PROTEINS; RESINS; SUBSTRATES

Citation Formats

Marcondes, Marcelo F., Torquato, Ricardo J.S., Assis, Diego M., Juliano, Maria A., Hayashi, Mirian A.F., and Oliveira, Vitor. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates. United States: N. p., 2010. Web. doi:10.1016/J.BBRC.2009.11.014.
Marcondes, Marcelo F., Torquato, Ricardo J.S., Assis, Diego M., Juliano, Maria A., Hayashi, Mirian A.F., & Oliveira, Vitor. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates. United States. https://doi.org/10.1016/J.BBRC.2009.11.014
Marcondes, Marcelo F., Torquato, Ricardo J.S., Assis, Diego M., Juliano, Maria A., Hayashi, Mirian A.F., and Oliveira, Vitor. 2010. "Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates". United States. https://doi.org/10.1016/J.BBRC.2009.11.014.
@article{osti_22199955,
title = {Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates},
author = {Marcondes, Marcelo F. and Torquato, Ricardo J.S. and Assis, Diego M. and Juliano, Maria A. and Hayashi, Mirian A.F. and Oliveira, Vitor},
abstractNote = {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.},
doi = {10.1016/J.BBRC.2009.11.014},
url = {https://www.osti.gov/biblio/22199955}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 1,
volume = 391,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 2010},
month = {Fri Jan 01 00:00:00 EST 2010}
}