High-level expression and purification of MyoD, myogenin, and E12.
The myogenic regulatory factors (MRFs), MyoD, myogenin, Myf-5, and MRF4, function as transcriptional activators of muscle-specific gene expression by forming heterodimers with the ubiquitously expressed products of the E2A gene, E12 and E47. To enable quantitative biochemical and biophysical analyses of the wild-type proteins, as well as mutants designed to reveal structure-function relationships, we developed protocols for the high-level expression and rapid purification of milligram quantities of MyoD, myogenin, and E12 using conventional biochemical techniques. T7 expression systems were used to direct expression of cDNA encoded proteins in Escherichia coli. Whereas MyoD and E12 were expressed well without alteration, high-level expression of myogenin required changing several rare arginine codons by in vitro mutagenesis to a commonly used E. coli arginine codon. Presumably, inefficient translation of the rare arginine codons inhibited high-level expression of myogenin in the original expression plasmid. Purification protocols are described which involve a simple strategy of cell lysis, ammonium sulfate precipitation, and ion-exchange chromatography. Using this approach, 20 to 50 mg of MyoD, myogenin, or E12 can be purified to 90-95% homogeneity from induced cell pellets in 1 day's time.
Maleki
S J
SJ
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Hurlburt
B K
BK
D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D017570:MyoD Protein / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018008:Myogenin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D017570:MyoD Protein; D018008:Myogenin; D011994:Recombinant Proteins; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors