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

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• 作者：Meenakshi Upreti and Pramod C. Rath
• 关键词：DNA binding activity ; interferon ; interferon regulatory factor ; 1 (IRF ; 1)
• 刊名：Molecular Biology Reports
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：32
• 期：2
• 页码：103-116
• 全文大小：512 KB

文摘

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 1029bp IRF-1 cDNA in pGEX-2TK expression vector in Escherichia coli XL-1 blue cells. Recombinant GST-IRF-1 was highly expressed as a 66kDa soluble protein by IPTG-induction, and was biologically active in terms of its DNA binding activity with a 24bp 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 25×- to 100×-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.