Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
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pALEX, a prokaryotic expression vector, was constructed in which the multiple cloning site (MCS, polylinker) is flanked by sequences encoding glutathione S-transferase (GST) at the 5' end and a His6 residue tag at the 3' end. Open reading frames cloned into this vector can direct production of fusion proteins with GST at their N terminus and a His6 tag at their C terminus. This allows for the purification of full-size fusion proteins by a sequential two-step procedure on glutathione-agarose and Ni(2+)-agarose columns.
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PMID:pALEX, a dual-tag prokaryotic expression vector for the purification of full-length proteins. 759 Mar 19

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.
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PMID:Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins. 840 15