کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1205205 965186 2008 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry
چکیده انگلیسی

Green fluorescent protein (GFP) fused to the C-terminal 100 amino acids of CAAT enhancer binding protein (C/EBP) also containing an N-terminal (His)6 tag (GFP-C/EBP) was used as a transcription factor model to test whether thiol-disulfide exchange reactions could be used to successfully purify transcription factors. A symmetrical dithiol oligonucleotide with dual CAAT elements was constructed with 5′ and 3′ thiols. Upon reduction, circular dichroism confirms it spontaneously anneals with its internally complementary sequence to form the hairpin structure:Figure optionsDownload as PowerPoint slideThe specific GFP-C/EBP protein–DNA complex, formed in solution at nM concentrations, could then be recovered (trapped) via thiol-disulfide exchange with a disulfide thiopropyl-Sepharose and eluted with dithiothreitol. GFP-C/EBP was isolated from crude bacterial extract treated with iodoacetamide; DNA binding by GFP-C/EBP was unaltered by carboxyamidomethylation. Eluted GFP-C/EBP was of high purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein, after in-gel digestion with trypsin, was also characterized by capillary reversed-phase liquid chromatography–nano-electrospray ionization-tandem mass spectrometry and the results analyzed using MASCOT software searching of the non-redundant protein database. A score of 1874 with a sequence coverage of 51% encompassing both termini and internal sequences for the match with GFP-C/EBP confirms its identity and sequence. The method has high potential for the identification and characterization of transcription factors and other DNA-binding proteins.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1202, Issue 1, 15 August 2008, Pages 75–82
نویسندگان
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