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. 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.
Rudert
F
F
Genesis Research and Development Corporation Limited, Auckland, New Zealand. frudert@genesis.co.nz
Visser
E
E
Gradl
G
G
Grandison
P
P
Shemshedini
L
L
Wang
Y
Y
Grierson
A
A
Watson
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism; D019014:fas Receptor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D004247:DNA; D005982:Glutathione Transferase; D013917:Thrombin