Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.
Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells.
Chumakov
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A
Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine 90048.
Koeffler
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HP
D017501:Adenovirus E1B Proteins / Q000378:metabolism*; D001483:Base Sequence; D002461:Cell Line, Transformed; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D017501:Adenovirus E1B Proteins; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D005982:Glutathione Transferase