Reconstitution of an E box-binding Myc:Max complex with recombinant full-length proteins expressed in Escherichia coli.
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.
Department of Biochemistry, University of California, Riverside, CA 92521, USA.
Anthony
A
Farina
Francesco
F
Faiola
Ernest
E
Martinez
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D003001:Cloning, Molecular; D019281:Dimerization; D024721:E-Box Elements / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D006801:Humans; D016350:Leucine Zippers / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C106465:MNT protein, human; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012097:Repressor Proteins