Expression and characterization of the human c-myc DNA-binding protein.

Abstract

In an effort to study in detail the nature of the protein product of the human protoorcogene 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 thermoind,pcible 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 tfo immunoprecipitate the p11$am--Yc 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 Burlitt 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 doublestranded DNA.

Max: functional domains and interaction with c-Myc.

“…2B), a previously described c-Myc DNA-binding sequence (Halazonetis and Kandil 1991). Full-length bacterially produced c-Myc protein was partially purified by conventional column chromatography as described (Watt et al 1985). The full-length c-Myc protein alone could not bind specifically to DNA alone, even at relatively high concentration (5 JJLM).…”

“…The modified pDS65-6xHis vector was modified further to include an Nsil site, and between the Clal and Nsil sites a max fragment was inserted from the upstream Taql site to the Nsil site from pVZlp21Max, a plasmid that encodes the shorter 151-aminoacid Max polypeptide (a gift from E. Blackwood and R. Eisenman), generating the new plasmid pDS-Max. The PRMyc expression vector that encodes full-length c-Myc (a gift from R. Watts) has been described previously (Watt et al 1985).…”

“…Full-length c-Myc protein was prepared and purified as described (Watt et al 1985) to a purity of -50% as estimated by gel electrophoresis (data not shown). The truncated c-Myc proteins and the Max protein were prepared and purified as described (Abate et al 1990).…”

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

“…The resulting plasmid pARDS18-3 produced a large amount of polypeptide (M.W., 60 kd) in E. coli BL21(DE3). These fusion polypeptides were purified by the procedures of Watt et al (1985) and used as the antigen to immunize rabbits as described previously (Hirano et al, 1988). dskl protein was produced in E. coli.…”

Production of a truncated human c‐myc protein which binds to DNA

“…The c-myc protein is localized in the nucleus [3], and is associated with the nuclear matrix [4]. The c-myc protein produced in E. coli exhibits a high sequence-nonspecific affinity for double-stranded DNA [5]. The human cmyc gene is composed of three exons, exons 2 and Correspondence address: Y. Kurosawa, Institute for Comprehensive Medical Science, Fujita-Gakuen Health University, Toyoake, Aichi 470-l 1, Japan…”