Direct purification of multiple ATF/E4TF3 polypeptides from HeLa cell crude nuclear extracts using DNA affinity latex particles.
We developed a method using affinity latex particles to rapidly and efficiently purify DNA-binding proteins directly from crude cell extracts. The particles are composed of a styrene core and a polyglycidyl methacrylate surface, to which DNA oligomers were immobilized by means of epoxy groups. Multiple polypeptides were copurified, which bound to the ATF/E4TF3-binding site from crude nuclear extracts of HeLa cells, within a few hours. Affinity-purified polypeptides stimulated transcription in vitro from a promoter in which ATF/E4TF3-binding sites were present. At least eight polypeptides with molecular masses of 116, 80, 65, 60, 55, 47, 45, and 43 kDa were copurified. About 2 micrograms of the 43-kDa protein was purified directly from 8 mg of crude nuclear extracts. All the polypeptides directly bound to the same DNA sequence and were thought to form a family. The results indicated that the particles are useful for quickly purifying various DNA-binding proteins directly from crude cell extracts.
Inomata
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Y
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Kawaguchi
H
H
Hiramoto
M
M
Wada
T
T
Handa
H
H
D051696:Activating Transcription Factors; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism*; D004852:Epoxy Compounds; D006367:HeLa Cells / Q000648:ultrastructure; D006801:Humans; D007840:Latex; D008689:Methacrylates; D008969:Molecular Sequence Data; D011137:Polystyrenes; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
D051696:Activating Transcription Factors; D001798:Blood Proteins; D004852:Epoxy Compounds; D007840:Latex; D008689:Methacrylates; D011137:Polystyrenes; D014157:Transcription Factors; D004247:DNA; C007870:glycidyl methacrylate