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

Concatemers, tandem copies of DNA elements ligated together, are widely use d for the DNA affinity chromatography of transcription factors. Purificatio n of different transcription factors using discrete, concatemeric and T-18: A(18) tailed DNA affinity columns was studied. Columns having a discrete DN A sequence bound by cytidylic-adenylic-adenylic-thymidylic oligonucleotide (CAAT) enhancer binding protein (C/EBP) yields significantly more green flu orescent protein-C/EBP (GFP-C/EBP) fusion protein than a concatemeric DNA c olumn made from five tandem repeats of the same DNA sequence. For lac repre ssor protein, the concatemeric and T-18:A(18) tailed columns show greater r etention times than a discrete, untailed DNA affinity column. It was observ ed that the T-18:A(18) tailed column gives better resolution than either th e discrete or concatemeric columns, of mixtures containing both lac repress or and GFP-C/EBP. Discrete concatemeric and T-18:A(18) tail columns all bou nd the Spl transcription factor and showed similar retention. The T-18: A(1 8) tailed column gives higher yield for Spl than the other columns. Our stu dy shows concatemeric columns do not have any distinct advantage for the th ree different transcription factors we studied including Spl, the original justification for the concatemeric approach. (C) 2001 Elsevier Science B.V. All rights reserved.