Strep-tag II for one-step affinity purification of active bHLHzip domain of human c-Myc.
The c-Myc protein, the product of the c-myc 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 C-terminal 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.
Zwicker
N
N
Hans-Knöll-Institute for Natural Products Research, Jena, Germany.
Adelhelm
K
K
Thiericke
R
R
Grabley
S
S
Hänel
F
F
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry; D005982:Glutathione Transferase / Q000302:isolation & purification; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers; D011487:Protein Conformation; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D019809:Streptavidin / Q000737:chemistry; D014157:Transcription Factors
D001425:Bacterial Outer Membrane Proteins; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C078167:OMPA outer membrane proteins; D019809:Streptavidin; D005982:Glutathione Transferase