Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein.
DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.
D017456:Adenovirus Early Proteins; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000276:immunology / Q000633:toxicity; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology
D017456:Adenovirus Early Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase