pLEF, a novel vector for expression of glutathione S-transferase fusion proteins in mammalian cells

An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purifica...

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Veröffentlicht in:	Gene 1996-03, Vol.169 (2), p.281-282
Hauptverfasser:	Rudert, Fritz, Visser, Elizabeth, Gradl, Gabriele, Grandison, Prudence, Shemshedini, Lirim, Wang, Yue, Grierson, Alastair, Watson, James
Format:	Artikel
Sprache:	eng
Schlagworte:	Amino Acid Sequence Animals apoptosis receptor Base Sequence Cell Line DNA fas Receptor - chemistry fas Receptor - genetics fas Receptor - isolation & purification Gene Expression - genetics Genetic Vectors - genetics Glutathione Transferase - genetics Humans Mammals Mice Molecular Sequence Data neomycin selection phosphorylation Protein purification Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Thrombin - metabolism
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Beschreibung
Zusammenfassung:	An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purification of GST::CD95IC from 32P-labelled L929 cells and cleavage with thrombin revealed that CD95IC was phosphorylated in vivo when produced as a GST fusion protein. Therefore, pLEF may facilitate the mapping of in vivo-modified sites of eukaryotic proteins
ISSN:	0378-1119 1879-0038
DOI:	10.1016/0378-1119(95)00820-9