Enteric adenovirus type 40: expression of E1B proteins in vitro and in vivo.
The genes encoding the enteric adenovirus type 40 E1B proteins designated 19K, 55K, and 15K (55K related) have been cloned into the pET3a expression vector and synthesized by in vitro transcription and translation and by in vivo expression after induction in bacteria. The 19K product expressed in bacteria is recognized by anti-peptide sera specific for the C-terminal region of the open reading frame and has the same M(r) as 19K protein immunoprecipitated from virus-infected cells. The 55K protein synthesized in bacteria is insoluble except under extreme denaturing conditions, but after in vitro transcription followed by translation, a polypeptide of the predicted size is obtained. The 15K protein, equivalent to the first 73 and last 29 of the 476-residue 55K protein with an internal deletion of 374 amino acids, is expressed to a high level in bacteria in a soluble form and interacts weakly but specifically with N- and C-terminal anti-peptide sera. The bacterially expressed 15K protein was used to raise antibodies in rabbits. This serum precipitates the 55K protein expressed by in vitro translation, but only the 15K product can be immunoprecipitated from virus-infected cells. The same antiserum, however, detects the 55K protein in infected cells by Western blotting, at a time broadly coinciding with the onset of DNA replication. This is the first identification of Ad40 55K protein in infected cells and confirms that the Ad40 22S mRNA can be utilized in vivo. The question of whether this protein is functional can now be addressed.
Bailey
A
A
Medical Research Council Virology Unit, Institute of Virology, Glasgow, Scotland.
MacKay
N
N
Mautner
V
V
D017501:Adenovirus E1B Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014158:Transcription, Genetic
D017501:Adenovirus E1B Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins