FOLDING AND PURIFICATION OF A RECOMBINANTLY EXPRESSED INTERFERON REGULATORY FACTOR, IRF-4

Abstract Interferon regulatory factor 4 (IRF-4), an intracellular, multidomain protein, is a member of the interferon regulatory factor family and a lymphoid-specific transcription factor that can form a ternary complex with DNA and the transcription factor PU.1. Recombinant human IRF-4 was expressed in Escherichia coli and purified from the soluble cell extract and the insoluble inclusion bodies. The inclusion bodies were solubilized with guanidinium-hydrochloride and sequentially buffer exchanged into urea- and then NaCl-containing solutions. This two-step process for the removal of the denaturants was the critical step to allow for the correct folding of IRF-4. Following purification through immobilized metal affinity, hydrophobic interaction, and gel permeation chromatographies, the renatured protein was shown to be structurally and physically equivalent to a sample of IRF-4 produced in the soluble fraction of E. coli cells. This was confirmed by near and far UV circular dichroism analysis, including thermal stability analysis. The purified IRF-4 was also shown to be capable of binding DNA in a PU.1-dependent manner by electrophoretic mobility shift analysis. The protein folding and purification methods are suitable for producing large quantities of full-length IRF-4.