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CD40 (human):COMP (human), (recombinant)

First non-antibody blocker of CD40L signalling
 
ALX-522-064-C010 10 µg 425.00 USD
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Product Specification

Alternative Name:TNFRSF 5
 
MW:~36kDa (SDS-PAGE).
 
Source:Produced in HEK 293 cells. The extracellular domain of human CD40 (aa 1-193) is fused to a FLAG®-tag at the N-terminus and to human cartilage oligomeric matrix protein (COMP, aa 32-80) at the C-terminus.
 
UniProt ID:P25942
 
Concentration:0.1mg/ml after reconstitution.
 
Formulation:Lyophilized. Contains PBS.
 
Purity:≥90% (SDS-PAGE)
 
Endotoxin Content:<0.1EU/µg purified protein (LAL test; Bio Whittaker).
 
Specificity:Binds human CD40L (CD154).
 
Reconstitution:Reconstitute with 100µl sterile distilled water. Further dilutions should be made with medium containing 5% fetal calf serum or a carrier protein.
 
Shipping:Shipped on Blue Ice
 
Long Term Storage:-20°C
 
Use/Stability:Stable for at least 6 months after receipt when stored at -20°C.
 
Handling:Avoid freeze/thaw cycles. After reconstitution, prepare aliquots and store at -20°C.
 
Scientific Background:COMP (Cartilage Oligomeric Matrix Protein) also designated thrombospondin 5 (TSP 5) is an oligomeric protein synthesized by chondrocytes in all type of cartilage. It belongs to the thrombospondin family of extracellular proteins. It is a pentameric molecule forming a structure similar to a bouquet of flowers. It may have storage and delivery function for hydrophobic cell-signalling molecules such as vitamin D.
COMP is a calcium-binding protein composed of 5 identical disulfide-linked subunits of 755 amino acids each with a high percentage of acidic residues (30%). The molecular weight of the homopentamer is 524 kDa, as determined by sedimentation equilibrium ultracentrifugation. Each subunit is composed of an N-terminal coiled-coil domain (unique to COMP), four epidermal growth factor (EGF)-type repeats, followed by eight calmodulin-like repeats and a globular C-terminal domain. The C-terminal domain binds to collagens I, II and IX.
Chimeras composed of proteins fused to the pentamerization domain of COMP (aa 32-75) show an increased avidity compared to the protein alone. Historical results have shown that COMP is a potent blocker of mouse CD40L-dependent B cell proliferation without the requirement of any crosslinking reagent (enhancer).
 
Technical Info/Product Notes:FLAG is a registered trademark of Sigma-Aldrich Co.
 
522-064

Method: rhCD40-COMP blocks rmsCD40L-induced proliferation of murine B cells. Splenocytes from Balb/c mice were purified on B220 magnetic beads using the MACS system. The resulting B cells were put into culture at a density of 70’000 cells/well in a 96-well plate. 500 ng/ml of rmsCD40L (Prod. No. ALX-522-043) in the presence of 1 mg/ml of enhancer (Prod. No. ALX-804-034) were preincubated for 30 min. at RT with the indicated concentrations of rhCD40-COMP (Prod. No. ALX-522-064) or rhCD40:Fc (Prod. No. ALX-522-016), and then added to the purified B cells. After 36 hours, 0.5mCi of 3H-Thymidine/well was added. Cells were pulsed for 10 hours, freeze-thawed, harvested and counted. Results are expressed as % of inhibition of the maximal proliferation yielded when no rhCD40-COMP or rhCD40:Fc were added.

comp structure
Figure: Domain structure of a COMP subunit (upper figure) and structure of the CD40:COMP chimera (lower figure).
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522-064 comp structure

Product Literature References

Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors: N. Holler, et al.; J. Immunol. Methods 237, 159 (2000), Abstract;

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