Expression and DNA binding activity of the recombinant interferon regulatory factor-1 (IRF-1) of mouse

Interferon regulatory factor- 1 ( IRF- 1) is a positive transcription factor for genes involved in immune response, cell growth regulation and apoptosis in mammalian cells. Many agents like virus, interferon ( IFN), double- stranded RNA ( dsRNA), proinflammatory cytokines, prolactin etc. induce IRF- 1 at transcriptional level. IRF- 1 transcriptionally activates many IRF- 1- regulated genes during normal physiological and pathological conditions. We have expressed recombinant mouse IRF- 1 ( 329 amino acids) as a GST( glutathione- S- transferase)- IRF- 1 fusion protein from a 1029 bp IRF- 1 cDNA in pGEX- 2TK expression vector in Escherichia coli XL- 1 blue cells. Recombinant GST- IRF- 1 was highly expressed as a similar to 66 kDa soluble protein by IPTG- induction, and was biologically active in terms of its DNA binding activity with a 24 bp specific oligonucleotide, i. e. P-32( GAAAGT) 4 but not with a similar but nonspecific oligonucleotide i. e. P-32( GAAA) (6). GST- alone expressed from the vector did not bind P-32( GAAAGT) (4). We observed multiple ( 1 - 4) GST- IRF- 1-( GAAAGT) 4 protein - DNA complexes which were competed out by 25 x- to 100 x- fold molar excess of ( GAAAGT) 4 showing that the complexes 1 - 4 were specific for IRF- 1. Such GAAANN ( N= any nucleotide) hexanucleotides occur in the promoters of many virus- and interferon-inducible mammalian genes. Multimeric GAAAGT/ C sequences are inducible by virus, IFN, dsRNA and IRF- 1. Specificity of DNA binding by IRF- 1 lies in the 5th and 6th nucleotides in the GAAAGT sequence. Multiple IRF- 1- DNA complexes should stimulate transcription by IRF- 1.