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 (WD40Apaf-1) in Escherichia coli. The WD40 domain expressed contains 825 amino acids in addition to an N-terminal His6 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 WD40Apaf-1 and cytochrome c. The binding interaction has been quantified by spectrophotometric titration of cytochrome c with recombinant WD40Apaf-1. The results demonstrate a weak binding for cytochrome c and WD40Apaf-1 interaction, the binding affinity being 390 nM. The analysis indicates a 2:1 or possibly even 3:1 stoichiometry for cytochrome c and WD40Apaf-1 binding interaction. © 2009 Elsevier Inc. All rights reserved.