Comparative studies on discrete and concatemeric DNA-sepharose columns for purification of transcription factors

Concatemers, tandem copies of DNA elements ligated together, are widely used for the DNA affinity chromatography of transcription factors. Purification of different transcription factors using discrete, concatemeric and T-18:A(18) tailed DNA affinity columns was studied. Columns having a discrete DNA sequence bound by cytidylic-adenylic-adenylic-thymidylic oligonucleotide (CAAT) enhancer binding protein (C/EBP) yields significantly more green fluorescent protein-C/EBP (GFP-C/EBP) fusion protein than a concatemeric DNA column made from five tandem repeats of the same DNA sequence. For lac repressor protein, the concatemeric and T-18:A(18) tailed columns show greater retention times than a discrete, untailed DNA affinity column. It was observed that the T-18:A(18) tailed column gives better resolution than either the discrete or concatemeric columns, of mixtures containing both lac repressor and GFP-C/EBP. Discrete concatemeric and T-18:A(18) tail columns all bound the Spl transcription factor and showed similar retention. The T-18: A(18) tailed column gives higher yield for Spl than the other columns. Our study shows concatemeric columns do not have any distinct advantage for the three different transcription factors we studied including Spl, the original justification for the concatemeric approach. (C) 2001 Elsevier Science B.V. All rights reserved.