Oligonucleotide trapping method for purification of transcription factors

Abstract

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 n M) 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 (3668- versus 1028-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.