Purification and biological characterization of an adenovirus type 2 E1A protein expressed in Escherichia coli.
The adenovirus 2 E1A gene encodes a multifunctional protein of 289 amino acids that can immortalize primary rodent cells and transcriptionally activate a number of viral and cellular genes. To facilitate an understanding of the molecular basis for the various actions of E1A, we have redesigned our bacterial expression vector (Ko, J.-L., and Harter, M. L. (1984) Mol. Cell. Biol. 4, 1427-1439) containing the cloned E1A gene such that a soluble authentic E1A protein now constitutes approximately 1.5% of the total Escherichia coli cellular protein. Further, we have developed a simple rapid purification scheme without the use of detergents or denaturants and show a purity of greater than 98% with a yield of approximately 53%. The E1A so purified is biologically active, stimulating cellular DNA synthesis following microinjection into quiescent NIH 3T3 and REF52 cells. In another report (Spangler, R., Bruner, M., Dalie, B., and Harter, M. L. (1987) Science 237, 1044-1046) we have also shown that our purified E1A protein activates transcription from appropriate promoters in an in vitro system.
Bruner
M
M
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
Dalie
B
B
Spangler
R
R
Harter
M L
ML
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic
D017456:Adenovirus Early Proteins; D000956:Antigens, Viral; D009856:Oncogene Proteins, Viral