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Stability and apoptotic activity of recombinant human cytochrome c.

An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.

Pubmed ID: 14680826

Authors

  • Olteanu A
  • Patel CN
  • Dedmon MM
  • Kennedy S
  • Linhoff MW
  • Minder CM
  • Potts PR
  • Deshmukh M
  • Pielak GJ

Journal

Biochemical and biophysical research communications

Publication Data

December 19, 2003

Associated Grants

  • Agency: NIEHS NIH HHS, Id: R21 ES10774

Mesh Terms

  • Animals
  • Apoptosis
  • Apoptotic Protease-Activating Factor 1
  • Caspase 3
  • Caspases
  • Cytochromes c
  • Enzyme Activation
  • Enzyme Stability
  • Escherichia coli
  • Horses
  • Humans
  • Models, Molecular
  • Protein Conformation
  • Protein Engineering
  • Proteins
  • Recombinant Proteins
  • Species Specificity