Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics.
Genome projects are identifying an ever-increasing number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products. Our goal was to develop a strategy to allow human single-chain variable fragment (scFv) antibodies to be used for these endeavors. A library containing 7x10(9) individual variants was displayed by bacteriophage and selected against a biotinylated peptide corresponding to the C-terminal 15 amino acid residues of Ku86, one component of a heterodimer involved in double-stranded DNA break repair. Four unique scFv antibodies were recovered that not only recognized the selected peptide, but also the intact protein. Three of the scFv antibodies were expressed in soluble form and recognized Ku86 by Western analysis. The affinity of one of the scFv antibodies for Ku86 was 16 nM as measured by BIAcore analysis. scFv immunoprecipitation of Ku86 also isolated the other component of the heterodimer, Ku70, as determined by Western analysis and mass spectrometry. These results demonstrate the utility of scFv antibodies as invaluable reagents for functional genomics.
Siegel
R W
RW
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA.
Allen
B
B
Pavlik
P
P
Marks
J D
JD
Bradbury
A
A
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D034961:Antigens, Nuclear; D019921:Biotinylation; D015153:Blotting, Western; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D016678:Genome; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D011233:Precipitin Tests; D011485:Protein Binding; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
D000906:Antibodies; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D019151:Peptide Library; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen