Purification of DNA-binding transcription factors by their selective adsorption on the affinity latex particles.
A simple method with the use of affinity latex particles has been developed for the fast and efficient purification of sequence-specific DNA-binding proteins on the basis of their ability to selectively bind to their target sequences. Complementary oligodeoxynucleotides that contained a recognition site for a sequence-specific DNA-binding protein were chemically synthesized, annealed and ligated to give oligomers. The oligomers were coupled to latex particles, composed of polyglycidyl methacrylate, using cyanogen bromide to yield affinity latex particles. The concentration of covalently bound DNA on the affinity latex particles was 6 times as much DNA per ml as that in the Sepharose resin conventionally used. By sequential batch-wise procedures with the affinity particles, one of the sequence-specific DNA binding transcription factors, ATF or E4TF3, was quickly and efficiently purified to homogeneity from either a protein fraction in which the factor was enriched or a crude cell extract.
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Japan.
H
H
Kawaguchi
A
A
Asai
Y
Y
Ohtsuka
H
H
Watanabe
T
T
Wada
H
H
Handa
D051696:Activating Transcription Factors; D000327:Adsorption; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D002498:Centrifugation; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D004247:DNA; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D051696:Activating Transcription Factors; D001798:Blood Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA; D003488:Cyanogen Bromide