Expression and Purification of Recombinant Human S100A8 and S100A9 for Characterization of Their Roles as Danger Signals

University essay from Lunds universitet/Tillämpad biokemi

Abstract: The direct involvement of S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) in numerous human diseases have been discovered more than 20 years ago, and they have ever since emerged as a very potent biomarker. Increased levels of S100A8 and S100A9 have been found in inflammation and various cancers. As complexes, they become crucial danger signals and act as endogenous activators of TLR4 (Toll-like receptor 4) and RAGE (Receptor for Advanced Glycation Endoproducts) on sentinel cells. As danger signals, they activate TLR4- and RAGE-dependent signaling cascades, especially the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signalling pathway. S100A8 and S100A9 have become the focus of many current researches since they seem to play a central role as danger signals, however the initial events during binding of the proteins to their receptors remain questionable. The aim of this project was to produce recombinant human S100A8 (rhS100A8) and recombinant human S100A8/S100A9 (rhS100A8/S100A9) to characterize their role as ligands of TLR4-MD2 and RAGE in vitro with surface plasmon resonance (SPR) and as activators of the NF-κB cell-signalling pathway in cell-based in vitro system with NF-κB Luciferase reporter assay. Within the scope of producing rhS00A8, the protein was expressed in its wild- type (wt) form and compared to S100A8 fused to a His6-tag (His6-rhS100A8). The study observed a much lower stability of wt-rhS100A8, with degradation when subject to potential oxidation. Characterization of His6-S100A8 and wt- rhS100A8/S100A9 confirmed: i) in vitro binding activities of His6-S100A8 to TLR4-MD2 and RAGE with SPR studies; ii) cell-based in vitro binding activities of wt-rhS100A8/S100A9 to TLR4 with NF-κB Luciferase reporter assay and iii) lower binding activities of heterocomplex compared to homocomplex in both previously mentioned systems.

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