| TSG-6 (TNF-stimulated gene 6, also TNFIP6) is a secreted, 35-39 kD group A member of the LINK-Module superfamily of proteins (1-4). Mouse TSG-6 is synthesized as a 275 amino acid (aa) precursor. It contains a 17 aa signal sequence and a 258 aa mature region (5, 6). The mature region has an N-terminal link module (aa 36-129) and a C-terminal CUB (C1s/C1r, urchin embryonic growth factor, BMP1) domain (aa 135-246). Link modules bind hyaluronan (HA) and participate in extracellular matrix (ECM) assembly (7). Mature mouse TSG-6 shares 97%, 94% and 94% aa identity with rat, human and dog TSG-6, respectively. Cells reported to express TGF-6 include activated fibroblasts, synoviocytes, chondrocytes, neutrophils, proximal tubular epithelium, bronchial epithelium, endothelium, and visceral plus vascular smooth muscle (2, 8). TSG-6 has multiple functions, many of which involve the ECM. It is suggested to stabilize HA-rich ECM. It does so by serving as an intermediary, or link, between the individual subunits of extracellular decameric pentraxin 3 and the surrounding hyaluronan matrix (9). It also provides structure and organization to hyaluronan. This is accomplished by a TSG-6 mediated transfer of an 80-85 kD protein subunit from IalphaI (inter-alpha-inhibitor) to HA. IalphaI is a four-component, 225 kD serine protease inhibitor. It contains a protease inhibitor subunit (bikunin), two independent, accompaning protein chains (HC1 and HC2), and a short chondroitin sulfate linking moiety. TSG-6 is a catalyst for the removal and transient binding of either HC chain. Each chain is subsequently transferred and covalently-linked to the surrounding HA. This provides substance and reinforcement to the ECM (1, 2, 10, 11, 12). This disassembly of IalphaI also leads to free bikunin, which in the free state becomes a potent inhibitor of serine proteases (8) Source: Human CD33 Signal Peptide (Met 1-Ala 16), Mouse TSG-6 (Trp 18-Leu 275), HHHHHH, A DNA sequence encoding the signal peptide of human CD33 was joined with the mature mouse TSG-6 with a 6X histidine tag at the C-terminus (Trp 18-Leu 275, Accession O08859). The chimeric protein was expressed in a mouse myeloma cell line, NS0. Molecular Mass: Based on N-terminal amino acid sequencing, the recombinant mouse TSG-6 starts at Trp 18 and has a calculated molecular mass of 29.8 kD. As a result of glycosylation, the recombinant protein migrates as an approximately 39 kD protein in SDS-PAGE under reducing conditions. Endotoxin Level: < 1.0 EU per 1 µg of protein as determined by the LAL method. Activity: Measured by its ability to bind biotinylated Hyaluronan in a functional ELISA type binding assay with an estimated KD < 1 nM. Reconstitution: It is recommended that sterile PBS be added to the vial to prepare a working stock solution of no less than 100ug/ml. The carrier-free protein should be used immediately upon reconstitution to avoid losses in activity due to non-specific binding to the inside surface of the vial. For long term storage as a dilute solution, a carrier protein (e.g. 0.1% HSA or BSA) should be added to the vial. Storage and Stability: Lyophilized samples are stable for up to twelve months from date of receipt at -20C. Upon reconstitution, this protein, in the presence of a carrier protein, can be stored under sterile conditions at 2-8 C for one month or at -20C in a manual defrost freezer for three months without detectable loss of activity. Avoid repeated freeze-thaw cycles. |
