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 the production of the adenomatous polyposis coli (APC) tumor suppressor protein. 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-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.
Wade
J D
JD
Howard Florey Institute, University of Melbourne, Victoria, Australia. j.wade@hfi.unimelb.edu.au
Catimel
B
B
Faux
M C
MC
Burgess
A W
AW
Nice
E
E
Otvos
L
L
D011125:Adenomatous Polyposis Coli / Q000378:metabolism*; D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003110:Colonic Neoplasms / Q000737:chemistry* / Q000628:therapy; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D011233:Precipitin Tests / Q000379:methods; D017433:Protein Structure, Secondary; D012996:Solutions / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014407:Tumor Cells, Cultured; D014867:Water / Q000737:chemistry
D025601:Adenomatous Polyposis Coli Protein; D015335:Molecular Probes; D010455:Peptides; D012996:Solutions; D014867:Water