Trapping of transcription factors with symmetrical DNA using thiol-disulfide exchange chemistry.
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: 5'-HS-GCAGATTGCGCAATCTGC 3'-HS-CGTCTAACGCGTTAGACG The 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.
Panda
Markandeswar
M
Department of Chemistry, University of Texas San Antonio, 1 University Circle, San Antonio, TX 78249, USA.
Jiang
Daifeng
D
Jarrett
Harry W
HW
D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA