Recombinant Pro-TNF-alpha Fusion Protein, CF

Tumor necrosis factor alpha (TNF-alpha), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism (1, 2). Human TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD) (3). Within the ECD, human TNF-alpha shares 97% aa sequence identity with rhesus and 71%-92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-alpha. TNF-alpha is produced by a wide variety of immune, epithelial, endothelial, and tumor cells (1, 2). TNF-alpha is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface (4). Cell surface TNF-alpha can induce the lysis of neighboring tumor cells and virus infected cells, and it can generate its own downstream cell signaling following ligation by soluble TNFR I (2, 5). Shedding of membrane bound TNF-alpha by TNF-alpha -converting-enzyme (TACE or ADAM17) releases the bioactive cytokine, a 55 kDa soluble trimer of the TNF-alpha extracellular domain (6-8). TNF-alpha binds the ubiquitous 55-60 kDa TNF RI (9, 10) and the hematopoietic cell-restricted 80 kDa TNF RII (11, 12), both of which are also expressed as homotrimers (1, 2, 13). Both type I and type II receptors bind TNF-alpha with comparable affinity (14), although only TNF RI contains a cytoplasmic death domain which triggers the activation of apoptosis. Soluble forms of both types of receptors are released and can neutralize the biological activity of TNF-alpha (15). TACE/ADAM17 cleaves the 26 kDa form at the Ala76-Val77 bond to produce the 17 kDa form (6, 16). ADAM10 processes the 26 kDa form at the same site as TACE (17). ADAM9 cleaves the 26 kDa form at alternative sites, Ala74-Glu75 and Ser79-Ser80 (18). The use of the recombinant Pro-TNF-alpha fusion protein as a protein substrate for Pro-TNF-alpha processing proteases has been tested with recombinant TACE/ADAM17 (R&D Systems, Catalog # 930-ADB), ADAM10 (Catalog # 936-AD and 946-AD), or ADAM9 (Catalog # 949-AD). The disappearance of the fusion protein (45 kDa) and the appearance of the mature TNF-alpha (17 kDa) were followed by Western blot analysis using an anti-human TNF-alpha polyclonal antibody (Catalog # AF210 or BAF210).