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 approximately 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. 32P(GAAAGT)4 but not with a similar but nonspecific oligonucleotide i.e. 32P(GAAA)6. GST-alone expressed from the vector did not bind 32P(GAAAGT)4. We observed multiple (1-4) GST-IRF-1-(GAAAGT)4 protein-DNA complexes which were competed out by 25x- to 100x-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.
Upreti
Meenakshi
M
Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Rath
Pramod C
PC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D005982:Glutathione Transferase; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins
D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase