Expression in Escherichia coli of the death domain of the human p55 tumor necrosis factor receptor.
The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects. The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD). Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E. coli. The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock. Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography. This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture. The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI. We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis.
De Wilde
G
G
Department of Molecular Biology, Gent University and Flanders Interuniversity Institute for Biotechnology, 9000 Gent, Belgium.
Mertens
N
N
Boone
E
E
De Vreese
B
B
Van Beeumen
J
J
Fiers
W
W
Haegeman
G
G
D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D017434:Protein Structure, Tertiary; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics
D015703:Antigens, CD; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins