HIGH-LEVEL EXPRESSION AND PURIFICATION OF MYOD, MYOGENIN, AND E12

The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF 4, function as transcriptional activators of muscle-specific gene expr ession by forming heterodimers with the ubiquitously expressed product s of the E2A gene, E12 and E47. To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants de signed to reveal structure-function relationships, we developed protoc ols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical t echniques. T7 expression systems were used to direct expression of cDN A encoded proteins in Escherichia coil. Whereas MyoD and E12 were expr essed well without alteration, high-level expression of myogenin requi red changing several rare arginine codons by in vitro mutagenesis to a commonly used E. coil arginine codon. Presumably, inefficient transla tion of the rare arginine codons inhibited high-level expression of my ogenin in the original expression plasmid. Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulf ate precipitation, and ion-exchange chromatography. Using this approac h, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% hom ogeneity from induced cell pellets in 1 day's time. (C) 1997 Academic Press.