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 coregulato rs of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interactio n between the respective DNA binding domains. To make possible a thoro ugh biochemical, biophysical, and structural characterization of the p roperties of myocyte enhancer factor (MEF) proteins and of their inter actions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-SC) was deve loped. A T7 expression system was used to produce in high yield in Esc herichia coli an N-terminal fragment of MEF-2C comprising both the MAD S box and the MEF domain. Purification by a single round of cation-exc hange chromatography on a Resource-S HPLC column at elevated pH afford ed an essentially pure protein. Recombinant MEF-2C(1-117) bound with h igh affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutat ions in this sequence that replaced adenines with thymine or vice vers a did not significantly alter the affinity for MEF-2C(1-117). The intr oduction of G-C pairs into the core of the MEF-site, however, dramatic ally increased the concentration of MEF-2C(1-117) needed for half maxi mal DNA binding. We propose an explanation of the DNA binding specific ity of MEF-2C based on the intrinsic bending properties of the unbound DNA. (C) 1997 Academic Press.