Oligonucleotide trapping method for purification of transcription factors.
A new oligonucleotide trapping method in which a decameric oligonucleotide (AC)5 coupled to Sepharose is used to trap a complex of a transcription factor and its corresponding specific DNA element is described. The concentration of DNA element used in the trapping method was very low (50 nM) and hence discouraged binding of nonspecific proteins. We have shown that this method gives higher purity for green fluorescent protein CAAT enhancer binding chimeric protein (GFP-C/EBP) than the biotin-avidin method. We have also shown that the oligonucleotide trapping method has a capacity close to 95% of the theoretical capacity, which is significantly greater than the 15% capacity obtained with conventional DNA affinity columns. The purity of GFP-C/EBP obtained using a low concentration of the oligonucleotide in our trapping method is three-fold higher (3,668- versus 1,028-fold) than that obtained by conventional DNA affinity chromatography and the yield was also higher (36% versus 24%). Highly purified transcription factor B3 is obtained from Xenopus egg crude extract using the oligonucleotide trapping method as the only purification.
Gadgil
Himanshu
H
Department of Biochemistry, University, of Tennessee, Memphis 38163, USA.
Jarrett
Harry W
HW
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D014157:Transcription Factors / Q000302:isolation & purification*; D014981:Xenopus
D022762:CCAAT-Enhancer-Binding Proteins; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins