Expression and characterization of the human c-myc DNA-binding protein.
In an effort to study in detail the nature of the protein product of the human protooncogene c-myc, we have expressed the gene at high levels in Escherichia coli. The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E. coli. Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoinducible expression system is very efficient. The product was relatively stable and accumulated to approximately 10% of total cellular protein. A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses. A high-titer polyclonal antiserum was raised against the pure protein and shown to immunoprecipitate the p110gag-myc fusion protein of MC-29-infected quail cells. This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burkitt lymphoma cell line. We conclude that this 64-kilodalton protein is the human c-myc gene product since the E. coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000. Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for double-stranded DNA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002051:Burkitt Lymphoma / Q000032:analysis; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis; D009857:Oncogenes; D011485:Protein Binding
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D004247:DNA