Overexpression and reactivation of binding activity of the recombinant CTF/NF-1 transcription factor.
The transcription factor CTF/NF-1 is a multifunctional cellular protein which participates in the expression of host and viral genes and in the replication of viral DNA. A procedure was developed to obtain recombinant CTF/NF-1 protein in order to help identify the binding sites of CTF/NF-1 protein in the regulatory region of viral sequences. Cloning and expression of the CTF/NF-1 gene downstream from a T7 RNA polymerase promoter resulted in an insoluble protein produced in Escherichia coli. Specific binding by the recombinant CTF/NF-1 protein to its cognate recognition site was obtained only after a denaturation and renaturation procedure. No special chromatography steps were required to obtain a rCTF/NF-1 preparation which could be used for our binding studies. Specific binding was detected with an NF-1 oligonucleotide and with viral DNA templates. Binding to the viral DNA sequences was identical to that previously published with biochemically purified cellular CTF/NF-1 protein.
Amemiya
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IGEN Inc., Rockville, Maryland 20852.
Durham
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Major
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D000260:Adenoviruses, Human / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007577:JC Virus / Q000235:genetics; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; C077305:CTF-1 transcription factor; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors