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 whi ch TNF exerts its effects. The signaling capacity largely depends on the pr esence of an intact C-terminal protein-protein interaction domain, a so-cal led death domain (DD). Here we report the expression and purification of th e human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TR X-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 mater ial appeared as multimers; it could be removed by selective precipitation a nd a combination of ion-exchange and size-exclusion chromatography. This pu rification protocol yielded 30 mg of purified, monomeric protein from 1 lit er of shake-flask culture. The purified TRX-DD was found to be functional a s 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. (C) 2001 Academic Press.