title: The adenovirus E1A 243R protein purified from Escherichia coli under nondenaturing conditions is found in association with dnaK.
abstract: The adenovirus E1A 243R protein immortalizes primary cells in culture and induces part of the phenotypes required for transformation. It has also been shown to interact with a number of cellular polypeptides, including the product of the retinoblastoma gene. To understand more fully the molecular activities of the E1A 243R protein in association with these proteins as well as its role in the processes of cellular growth, we have developed a method for rapidly purifying this protein from genetically engineered Escherichia coli under nondenaturing conditions. The plasmid-encoded E1A protein, when expressed in a protease-deficient mutant, is found to have the same length and amino acid sequence as that which is produced in a mammalian cell. The procedure for purifying the E1A 243R protein from bacteria relies primarily upon immunoaffinity chromatography and the use of a peptide comprising the epitope recognized by an E1A-specific antibody. Elution of the E1A protein under this condition allows for gentle isolation and a purity that ranges from 90 to 96%. However, without the addition of micromolar amounts of ATP prior to its elution from the antibody column, the E1A protein is found in association with an E. coli protein of 70 kDa. Immunoblot analysis with a specific antibody showed that this bacterial protein was the heat shock protein dnaK, which is known to have extensive homology with the hsp-hsc70 family of proteins in mammalian cells. Recognition of E1A by the dnaK protein may very well reflect a situation that also occurs between the mammalian heat shock proteins and the E1A 243R protein after adenovirus infection.
authors.affiliation: Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5285.
authors.forename: D M
authors.initials: DM
authors.lastname: Wang
authors.forename: B
authors.initials: B
authors.lastname: Dalie
authors.forename: M L
authors.initials: ML
authors.lastname: Harter
mesh_terms: D017500:Adenovirus E1A Proteins / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D017500:Adenovirus E1A Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; C063842:dnaK protein, E coli