title: Bacterially expressed recombinant WD40 domain of human Apaf-1.
abstract: The apoptotic protease activating factor (Apaf-1) is a protein that binds to cytochrome c, and in the presence of dATP/ATP oligomerizes to assume the role of an adaptor platform for activating the caspase-9 zymogen. In order to study the biochemical and structural details of Apaf-1 function, we have generated an expression construct from pcDNA 3-Apaf-1XL for production of the WD40 domain ((WD40)Apaf-1) in Escherichia coli. The WD40 domain expressed contains 825 amino acids in addition to an N-terminal His(6) tag derived from the cloning vector. The expressed protein is invariably localized in the inclusion body fraction of E. coli. A simple protocol involving Sephadex G100 chromatography developed for purifying the protein starting from inclusion bodies has allowed protein recovery in highly pure form. Basic fluorescence and CD spectra indicate that the refolded protein has extensive secondary and tertiary structures. Immunoprecipitation studies have provided qualitative information about the binding interaction of (WD40)Apaf-1 and cytochrome c. The binding interaction has been quantified by spectrophotometric titration of cytochrome c with recombinant (WD40)Apaf-1. The results demonstrate a weak binding for cytochrome c and (WD40)Apaf-1 interaction, the binding affinity being 390 nM. The analysis indicates a 2:1 or possibly even 3:1 stoichiometry for cytochrome c and (WD40)Apaf-1 binding interaction.
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: P Nageswara
authors.initials: PN
authors.lastname: Rao
authors.forename: Damodar
authors.initials: D
authors.lastname: Gullipalli
authors.forename: Abani K
authors.initials: AK
authors.lastname: Bhuyan
mesh_terms: D053477:Apoptotic Protease-Activating Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D045304:Cytochromes c / Q000378:metabolism; D003911:Dextrans; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry; D013058:Mass Spectrometry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003911:Dextrans; D011993:Recombinant Fusion Proteins; D045304:Cytochromes c; C025614:sephadex; D004229:Dithiothreitol