Strep-tag® II for One-Step Affinity Purification of Active bHLHzip Domain of Human c-Myc
| Autor: | K Adelhelm, F Hänel, S Grabley, N Zwicker, R Thiericke |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Leucine zipper
Protein Conformation Recombinant Fusion Proteins Basic helix-loop-helix leucine zipper transcription factors Peptide Biology Chromatography Affinity General Biochemistry Genetics and Molecular Biology Proto-Oncogene Proteins c-myc Protein structure Affinity chromatography Humans Glutathione Transferase chemistry.chemical_classification Leucine Zippers Basic helix-loop-helix Basic Helix-Loop-Helix Leucine Zipper Transcription Factors Helix-Loop-Helix Motifs Fusion protein DNA-Binding Proteins Basic-Leucine Zipper Transcription Factors chemistry Biochemistry Phosphoprotein Streptavidin Bacterial Outer Membrane Proteins Transcription Factors Biotechnology |
| Zdroj: | BioTechniques. 27:368-375 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/99272rr05 |
| Popis: | The c-Myc protein, the product of the cmyc protooncogene, is a nuclear phosphoprotein with DNA-binding properties when heterodimerized with the Max protein. It contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix leucine zipper (bHLHzip) domain that directs heterodimerization and promotes DNA binding. Here, we describe the isolation of the bHLHzip domain of human c-Myc with a technique for efficient single-step purification. Using a Cterminal Strep-tag® II affinity peptide and a novel Streptactin-Sepharose® matrix, elution is performed under mild conditions by competition with the biotin analog desthiobiotin. No significant influence of the affinity tag on the activity of the bHLHzip domain was observed when the fusion protein was subjected to glutathione S-transferase (GST) pull-down assays for investigating its in vitro-binding properties with GST-Max. The use of the C-terminal Strep-tag II was shown to be more suitable for obtaining pure product fractions than use of the N-terminal GST affinity tag. |
| Databáze: | OpenAIRE |
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