Folding and purification of a recombinantly expressed interferon regulatory factor, IRF-4.
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.
Moellering
B J
BJ
Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1789, USA.
Yoshinaga
S K
SK
Hui
A
A
Delaney
J M
JM
Hara
S
S
Narhi
L O
LO
Westcott
K R
KR
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007372:Interferons / Q000378:metabolism; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D007372:Interferons