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.
Olteanu
Alina
A
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
Patel
Chetan N
CN
Dedmon
Matthew M
MM
Kennedy
Scott
S
Linhoff
Michael W
MW
Minder
Camille M
CM
Potts
Patrick R
PR
Deshmukh
Mohanish
M
Pielak
Gary J
GJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006736:Horses; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013045:Species Specificity
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D011994:Recombinant Proteins; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases