Cycled DNA immunoprecipitation procedure to enrich the target sequences for DNA binding proteins with the fold purification monitored.
Using centromere DNA binding protein (CENP-B) expressed as a fusion to beta-galactosidase in Escherichia coli, we established a cycled DNA immunoprecipitation procedure for enriching CENP-B binding sequences and monitoring the enrichment process. Degenerated synthetic oligonucleotides for an authentic CENP-B binding sequence, inserted into a pUC-derived vector, were incubated with the crude CENP-B extract. DNA-protein complexes formed in vitro were immunologically precipitated utilizing the beta-galactosidase moiety as a tagged antigen. The effectiveness of repeating cycles of immunoprecipitation was demonstrated by the color selection method designed for pUC-derived plasmids, after introducing the precipitated plasmids into Escherichia coli. After three cycles of DNA immunoprecipitation, only a few kinds of sequences constituted the majority. By repeating two more cycles, the most predominant sequence was finally enriched until homogeneous, indicating the enrichment of the binding sequences in a hierarchical order. Further application to human genomic DNA showed that two EcoRI DNA fragments, 0.49 and 0.78 kb in size, were exclusively identified. This procedure can be applied to the systematic analysis of binding sequences for any other DNA binding proteins without production of any specific antibodies or further purification.
Sugimoto
K
K
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Japan.
Wakisaka
E
E
Himeno
M
M
D000906:Antibodies; D000941:Antigens / Q000235:genetics; D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D002503:Centromere / Q000737:chemistry; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism; D004270:DNA, Circular / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase