Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli

Protein Expr Purif. 2004 Apr;34(2):215-22. doi: 10.1016/j.pep.2003.11.021.


The c-Myc oncoprotein (Myc) is a DNA sequence-specific transcription factor that regulates transcription of a wide variety of genes involved in the control of cell growth, proliferation, differentiation, and apoptosis and its deregulated expression is implicated in many types of human cancer. Myc has an N-terminal transcription activation domain (TAD) that interacts with various coactivators and a C-terminal basic-helix-loop-helix-leucine zipper (bHLHZip) domain required for E box-specific DNA-binding and heterodimerization with its obligatory bHLHZip protein partner Max. The analysis of the mechanisms by which the Myc:Max complex regulates transcription at the molecular level in vitro has been hampered by the difficulty in obtaining highly pure recombinant Myc:Max heterodimers that contain full-length Myc with its complete TAD domain and that have sequence-specific DNA-binding activity. Here, we describe a simple method to reconstitute recombinant Myc:Max complexes from highly purified full-length proteins expressed in Escherichia coli that are soluble and highly active in E box-specific DNA-binding in vitro. The reconstituted Myc:Max complexes are stable and lack Max:Max homodimers. This procedure should facilitate the characterization of the DNA-binding and transcription activation functions of full-length Myc:Max complexes in vitro and in particular the role of Myc TAD-interacting cofactors and Myc:Max post-translational modifications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Cloning, Molecular
  • Dimerization
  • E-Box Elements / genetics*
  • Escherichia coli / genetics
  • Helix-Loop-Helix Motifs / genetics*
  • Humans
  • Leucine Zippers / genetics*
  • Plasmids / genetics
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / isolation & purification
  • Proto-Oncogene Proteins c-myc / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / isolation & purification
  • Repressor Proteins / metabolism*
  • Transcriptional Activation / genetics


  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • MNT protein, human
  • Proto-Oncogene Proteins c-myc
  • Recombinant Proteins
  • Repressor Proteins