Recombinant Human TLR4/MD2 Complex Protein

• Reactivity: Hu
• Applications: Bioactivity

Recombinant Human TLR4/MD2 Complex Protein Summary

• Details of Functionality: Measured by its ability to inhibit LPS-induced TNF-alpha secretion by PMA-differentiated U937 human histiocytic lymphoma cells. 20 µg/mL of recombinant human TLR4/MD-2 will inhibit >60% of the TNF-alpha secretion induced by LPS.
• Source: Mouse myeloma cell line, NS0-derived human TLR4/MD2 Complex protein
  

  Human TLR4 (Glu24-Lys631) Accession # O00206.2	S	10-His tag
  Human MD-2 (Glu17-Asn160) Accession # Q9Y6Y9.1	IEGRGGGSGGGSGGGS	10-His tag
  N-terminus		C-terminus

• Accession #: O00206.2 (TLR4) & Q9Y6Y9.1 (MD-2)
• N-terminal Sequence: Glu24 (TLR4) & Glu17 (MD-2)
• Protein/Peptide Type: Recombinant Proteins
• Gene: TLR4
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 70.6 kDa (TLR4), 19.2 kDa (MD-2).
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 95 kDa and 34 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human TLR4/MD2 Complex Protein

• ARMD10;CD284;ESOP-1;lipopolysaccharide response;Ly-96;Lymphocyte antigen 96;myeloid differentiation factor-2;myeloid differentiation protein-2;Protein MD-2;TLR-4;TOLL;Toll-like receptor 4
• TLR4/MD2 Complex
• TLR4/MD-2 Complex

Background

TLR4 is a 100 kDa type I transmembrane glycoprotein that belongs to the mammalian Toll-Like Receptor family of pathogen pattern recognition molecules. MD‑2, also known as ESOP-1, is a 25 kDa secreted protein that is required for TLR4‑mediated responses to bacterial lipopolysaccharide (LPS) (1 - 3). The human TLR4 cDNA encodes an 839 amino acid (aa) precursor that contains a 23 aa signal sequence, a 608 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 187 aa cytoplasmic domain. TLR4 contains 21 leucine rich repeats in its ECD and one cytoplasmic Toll/IL-1 receptor (TIR) domain (4). The ECD of human TLR4 shares approximately 25% aa sequence identity with other TLRs and 60% - 74% aa sequence identity with bovine, equine, feline, mouse, rat, and porcine TLR4. The human MD‑2 cDNA encodes a 160 aa precursor with an 18 aa signal sequence (5). Human MD‑2 shares 20% aa sequence identity with MD‑1 and 62% - 64% aa sequence identity with bovine, mouse, and rat MD‑2. MD‑2 associates with TLR4 on monocytes, macrophages, dendritic cells, and B cells (5 - 7). MD2 expression is required for cell surface localization of TLR4 and for optimal LPS‑induced TLR4 signaling (7, 8). MD‑2 also forms soluble disulfide-linked homo-oligomers which can interact with TLR4 (6). Through a domain separate from its TLR4-binding domain, MD‑2 extracts LPS from circulating CD14‑LPS complexes and carries the LPS into a ternary complex with TLR4 (9 - 11). The interaction of MD‑2/LPS with TLR4 induces receptor oligomerization and the triggering of an inflammatory response (12). Increased levels of plasma MD‑2 in septic shock patients sensitizes MD‑2 non-expressing epithelial cells to LPS and promotes widespread tissue inflammation (13).

1. Gangloff, M. and N.J. Gay (2004) Trends Biochem. Sci. 29:294.
2. Palsson-McDermott, E.M. and L.A. O’Neill (2004) Immunology 113:153.
3. Miyake, K. (2004) Semin. Immunol. 16:11.
4. Medzhitov, R. et al. (1997) Nature 388:394.
5. Shimazu, R. et al. (1999) J. Exp. Med. 189:1777.
6. Visintin, A. et al. (2001) Proc. Natl. Acad. Sci. USA 98:12156.
7. Akashi, S. et al. (2000) J. Immunol. 164:3471.
8. Nagai, Y. et al. (2002) Nat. Immunol. 3:667.
9. Re, F. and J.L. Strominger (2003) J. Immunol. 171:5272.
10. Kennedy, M.N. et al. (2004) J. Biol. Chem. 279:34698.
11. Gioannini, T.L. et al. (2004) Proc. Natl. Acad. Sci. USA 101:4186.
12. Saitoh, S. et al. (2004) J. Endotoxin Res. 10:257.
13. Pugin, J. et al. (2004) Blood 104:4071.

Publications for TLR4/MD-2 Complex (3146-TM)(23)

Publications using 3146-TM

• Khameneh, HJ;Bolis, M;Ventura, PMO;Cassanmagnago, GA;Fischer, BA;Zenobi, A;Guerra, J;Buzzago, I;Bernasconi, M;Zaman, GJR;Rinaldi, A;Moro, SG;Sallusto, F;Baulier, E;Pasquali, C;Guarda, G; The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells Mucosal immunology 2024-03-04 [PMID: 38447907] (Bioassay, N/A)
• Lee, H;Kim, B;Kim, M;Yoo, S;Lee, J;Hwang, E;Kim, Y; Characterization of the Antimicrobial Activities of Trichoplusia ni Cecropin A as a High-Potency Therapeutic against Colistin-Resistant Escherichia coli Pharmaceutics 2023-06-16 [PMID: 37376200] (Surface Plasmon Resonance, Human)
• X Wang, M Mayorga-Fl, KG Bien, AO Bailey, J Iwahara DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein The Journal of Biological Chemistry, 2022-10-08;0(0):102577. 2022-10-08 [PMID: 36220391] (Bioassay, Human)
• HL Xu, GH Chen, YT Wu, LP Xie, ZB Tan, B Liu, HJ Fan, HM Chen, GQ Huang, M Liu, YC Zhou Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway Journal of Ginseng Research, 2021-06-05;46(1):156-166. 2021-06-05 [PMID: 35058732] (Surface Plasmon Resonance, Human)
• D Tsubokawa, T Kikuchi, JM Lee, T Kusakabe, Y Yamamoto, H Maruyama Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration PloS Pathogens, 2021-06-03;17(6):e1009649. 2021-06-03 [PMID: 34081755] (Bioassay, Human)
• A Iannucci, V Caneparo, S Raviola, I Debernardi, D Colangelo, R Miggiano, G Griffante, S Landolfo, M Gariglio, M De Andrea Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding PLoS Pathog., 2020-09-09;16(9):e1008811. 2020-09-09 [PMID: 32903274] (Surface Plasmon Resonance, Human)
• E Jaeckle, E Brauchle, N Nottrodt, M Wehner, R Lensing, A Gillner, K Schenke-La, M Bach, A Burger-Ken Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy J. Biotechnol., 2020-09-06;0(0):. 2020-09-06 [PMID: 32898625] (Western Blot, Hamster)
• E Bahraoui, M Serrero, R Planès HIV-1 Tat - TLR4/MD2 interaction drives the expression of IDO-1 in monocytes derived dendritic cells through NF-kappaB dependent pathway Sci Rep, 2020-05-18;10(1):8177. 2020-05-18 [PMID: 32424165] (Bioassay, ELISA Capture, Human)
• A Kasus-Jaco, CA Land, AJ Stock, JL Washburn, HA Pereira Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9 Invest. Ophthalmol. Vis. Sci., 2020-04-09;61(4):16. 2020-04-09 [PMID: 32298435] (ELISA, Human)
• T Vogl, A Stratis, V Wixler, T Völler, S Thurainaya, SK Jorch, S Zenker, A Dreiling, D Chakrabort, M Fröhling, P Paruzel, C Wehmeyer, S Hermann, O Papantonop, C Geyer, K Loser, M Schäfers, S Ludwig, M Stoll, T Leanderson, JL Schultze, S König, T Pap, J Roth Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation J. Clin. Invest., 2018-04-03;0(0):. 2018-04-03 [PMID: 29611822] (Bioassay, Human)
• S Sun, M He, Y Wang, H Yang, Y Al-Abed Folic acid derived-P5779 mimetics regulate DAMP-mediated inflammation through disruption of HMGB1:TLR4:MD-2 axes PLoS ONE, 2018-02-15;13(2):e0193028. 2018-02-15 [PMID: 29447234] (Bioassay)
• J Honegr, R Dolezal, D Malinak, M Benkova, O Soukup, JSFD Almeida, TCC Franca, K Kuca, R Prymula Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery Molecules, 2018-01-04;23(1):. 2018-01-04 [PMID: 29300367] (Bioassay)
• P Durai, HJ Shin, A Achek, HK Kwon, RG Govindaraj, S Panneersel, D Yesudhas, J Choi, KT No, S Choi Toll-like Receptor 2 Antagonists Identified through Virtual Screening and Experimental Validation FEBS J., 2017-07-02;0(0):. 2017-07-02 [PMID: 28570013] (Surface Plasmon Resonance)
• Westman J, Papareddy P, Dahlgren M, Chakrakodi B, Norrby-Teglund A, Smeds E, Linder A, Morgelin M, Johansson-Lindbom B, Egesten A, Herwald H Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo. PLoS Pathog, 2015-12-08;11(12):e1005319. 2015-12-08 [PMID: 26646682] (Surface Plasmon Resonance)
• Loyau J, Malinge P, Daubeuf B, Shang L, Elson G, Kosco-Vilbois M, Fischer N, Rousseau F Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcgamma receptors. MAbs, 2014-01-01;6(6):1621-30. 2014-01-01 [PMID: 25484053] (Surface Plasmon Resonance, Human)
• Jung I, Shin S, Lee M, Kang T, Han H, Lee S, Kim W, Kim H, Park W, Kim H, Yun C, Lee E, Wu T, Park Y Enhancement of tumor-specific T cell-mediated immunity in dendritic cell-based vaccines by Mycobacterium tuberculosis heat shock protein X. J Immunol, 2014-07-02;193(3):1233-45. 2014-07-02 [PMID: 24990079] (Bioassay)
• Shang L, Daubeuf B, Triantafilou M, Olden R, Depis F, Raby A, Herren S, Dos Santos A, Malinge P, Dunn-Siegrist I, Benmkaddem S, Geinoz A, Magistrelli G, Rousseau F, Buatois V, Salgado-Pires S, Reith W, Monteiro R, Pugin J, Leger O, Ferlin W, Kosco-Vilbois M, Triantafilou K, Elson G Selective antibody intervention of Toll-like receptor 4 activation through Fc gamma receptor tethering. J Biol Chem, 2014-04-15;289(22):15309-18. 2014-04-15 [PMID: 24737331] (Bioassay, Human)
• Chen , Yu-Fon, Shiau , Ai-Li, Wang , Sheng-Hu, Yang , Jai-Sing, Chang , Sue-Joan, Wu , Chao-Lia, Wu , Tian-Shu Zhankuic acid A isolated from Taiwanofungus camphoratus is a novel selective TLR4/MD-2 antagonist with anti-inflammatory properties. J Immunol, 2014-02-14;192(6):2778-86. 2014-02-14 [PMID: 24532584] (Bioassay, Bacteria - Taiwanofungus camphoratus)
• Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med, 2012-07-29;18(0):1279. 2012-07-29 [PMID: 22842477] (Bioassay, Human)
• Feng C, Stamatos NM, Dragan AI, Medvedev A, Whitford M, Zhang L, Song C, Rallabhandi P, Cole L, Nhu QM, Vogel SN, Geddes CD, Cross AS Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex. PLoS ONE, 2012-04-09;7(4):e32359. 2012-04-09 [PMID: 22496731] (Bioassay, Human)
• Sakaguchi M, Murata H, Yamamoto K, Ono T, Sakaguchi Y, Motoyama A, Hibino T, Kataoka K, Huh NH TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding. PLoS ONE, 2011-08-01;6(8):e23132. 2011-08-01 [PMID: 21829704] (Bioassay, Human)
• Chambers MA, Whelan AO, Spallek R Non-acylated Mycobacterium bovis glycoprotein MPB83 binds to TLR1/2 and stimulates production of matrix metalloproteinase 9. Biochem. Biophys. Res. Commun., 2010-08-26;400(3):403-8. 2010-08-26 [PMID: 20800577] (Surface Plasmon Resonance, Bacteria)
• Zunt SL, Burton LV, Goldblatt LI, Dobbins EE, Srinivasan M Soluble forms of Toll-like receptor 4 are present in human saliva and modulate tumour necrosis factor-alpha secretion by macrophage-like cells. Clin. Exp. Immunol., 2009-03-09;156(2):285-93. 2009-03-09 [PMID: 19292767] (Western Blot)

Bioinformatics

• Gene Symbol: TLR4
• Uniprot:
  • O00206.2 (TLR4) & Q9Y6Y9.1 (MD-2)()