Bi-column method for purification of transcription factors

A novel bi-column method for purifying transcription factors, using two dif ferent columns and two different elution strategies is described. Lac repre ssor elutes at lower heparin concentrations from a lower affinity lactose o perator1 (Op1)-Sepharose column than from a higher affinity column containi ng the same sequence with a T18:A18 tail (Op1T(18)). A hi-column method was developed in which lac repressor fusion protein is eluted from the Op1-Sep harose with a low heparin concentration and trapped on a Op1T(18)-Sepharose column because of its higher affinity for the lac repressor protein. Eluti on of the latter column with buffer containing a high salt concentration gi ves significantly purer transcription factor than the conventionally used s ingle column methods and removes residual heparin. Highly pure CAAT enhance r binding protein and the B3 transcription factor are also obtained by usin g variants of this hi-column method. (C) 2001 Elsevier Science B.V. All rig hts reserved.