Folding and purification of a recombinantly expressed interferon regulatory factor, IRF-4

Interferon regulatory factor 4 (IRF-4), an intracellular, multidomain prote in, is a member of the interferon regulatory factor family and a lymphoid-s pecific transcription factor that can form a ternary complex with DNA and t he transcription factor PU.1. Recombinant human IRF-4 was expressed in Esch erichia coli and purified from the soluble cell extract and the insoluble i nclusion bodies. The inclusion bodies were solubilized with guanidinium-hyd rochloride and sequentially buffer exchanged into urea- and then NaCl-conta ining solutions. This two-step process for the removal of the denaturants w as the critical step to allow for the correct folding of IRF-4. Following p urification through immobilized metal affinity, hydrophobic interaction, an d gel permeation chromatographies, the renatured protein was shown to be st ructurally and physically equivalent to a sample of IRF-4 produced in the s oluble fraction of E. coli cells. This was confirmed by near and far UV cir cular dichroism analysis, including thermal stability analysis. The purifie d IRF-4 was also shown to be capable of binding DNA in a PU.1-dependent man ner by electrophoretic mobility shift analysis. The protein folding and pur ification methods are suitable for producing large quantities of full-lengt h IRF-4. (C) 1999 Academic Press.