High level expression in soluble form, one step purification, and characterization of the DNA binding domain of MEF-2C.
Members of the MEF-2 family of transcription factors act as coregulators of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains. To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed. A T7 expression system was used to produce in high yield in Escherichia coli an N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain. Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein. Recombinant MEF-2C (1-117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1-117). The introduction of G-C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1-117) needed for half maximal DNA binding. We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.
Meierhans
D
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Department of Chemistry, ETH-Zurich, Switzerland.
Allemann
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D004247:DNA; D004926:Escherichia coli; D018257:Helix-Loop-Helix Motifs; D064326:MEF2 Transcription Factors; D051379:Mice; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D018006:Myogenic Regulatory Factors; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA