Oligonucleotide trapping method for transcription factor purification systematic optimization using electrophoretic mobility shift assay

Abstract

Oligonucleotide trapping, where a transcription factor–DNA response element complex is formed in solution and then recovered (trapped) on a column, was optimized for the purification of CAAT/enhancer binding protein (C/EBP) from rat liver nuclear extract. Electrophoretic mobility shift assays (EMSAs) with ACEP24(GT) 5 oligonucleotide, containing the CAAT element, was used to estimate the binding affinity and concentration of C/EBP in the nuclear extract and then low concentrations of protein and oligonucleotide, which favor specific binding, were used for all further experiments. Also using EMSA, the highest concentrations of competitors, which inhibit non-specific binding but do not inhibit oligonucleotide binding by C/EBP, were determined to be 932 nM T 18 (single-stranded DNA), 50 ng/ml heparin (non-DNA competitor), and 50 μg/ml poly(dI:dC) (duplex DNA). Inclusion of 0.1% Tween-20 improved DNA binding. For complex formation, 110 μg nuclear extract was diluted to 0.2 nM C/EBP (apparent K d of C/EBP) and 1.34 nM ACEP24(GT) 5 was added, along with Tween-20 and the competitors. After incubation, the complex was trapped by annealing the (GT) 5 tail of the C/EBP–[ACEP24(GT) 5] complex to an (AC) 5–Sepharose column under flow at 4 °C. The column was washed with 0.4 M NaCl and the protein eluted with 1.2 M NaCl. The purification typically resulted in two proteins of apparent molecular mass 32 000 and 38 000. The smaller one, the major product, was identified to be C/EBP-α. The yield was 2.1 μg (66 pmol) of purified C/EBP-α p32. This systematic approach to oligonucleotide trapping is generally applicable for the purification of other transcription factors.