Structural and biosensor analyses of a synthetic biotinylated peptide probe for the isolation of adenomatous polyposis coli tumor suppressor protein complexes

Large numbers of colon tumors stem from mutations in the gene coding for th e production of the adenomatous polyposis coli (APC) tumor suppressor prote in. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp(1)) that dimer izes preferentially with this domain. We have undertaken the chemical synth esis of a modified form of this peptide (anti-APCp(2)) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis stud ies. The peptide was subjected to comprehensive chemical characterization t o confirm its purity. Secondary structural analysis by circular dichroism s pectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upo n the precise microenvironment. Significantly, a stable chi -helical struct ure was generated when the solvent conditions supported intramolecular salt -bridge formation along the helix barrel. The biotinylated anti-APCp(2) was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experi ment, injection of colonic cell lysate extracts (LlM1215) onto a size-exclu sion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the. immobilized anti-APCp(2) o n the biosensor surface. In another experiment, a high molecular mass prote in (M-r> 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp(2) peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp( 2) peptide and native APC and highlights the potential use of the former pe ptide in a multidimensional micropreparative chromatographic/biosensor/prot eomic protocol for the purification of APC alone and APC complexed with dif ferent biopolymers in various cell lines, and stages of tumor development.