The aryl hydrocarbon receptor (AhR) is a very unstable protein. AhR binds to the molecular chaperone complex (HSP90-p23-XAP2) to maintain a stable structure in the cytoplasm. After binding to ligands, such as dioxin, AhR translocates from the cytoplasm to the nucleus with a molecular chaperone complex. The protein forms a heterodimer with Arnt after nuclear transfer, functions as a transcription factor by binding to a xenobiotic responsive element (XRE), and induces the cytochrome P450 1A1 (CYP1A1). Because of the unstable protein, expression of the full-length AhR in the E. coli expression system is very difficult. Many studies investigated AhR using AhR domains in vitro. We expressed and purified the human full-length AhR in E. coli expression system. Furthermore, specific antibodies were prepared. Purified full-length AhR could bind to ligand. In the presence of ligand, α-helix and random coil of AhR increased and β-sheet decreased on CD spectrum. Full-length AhR could bind to HSP90, XAP2 and p23 in the presence or absence of ligand. We now show the biochemical properties of full-length AhR.
Cytokines play an important role in maintaining the homeostasis of hematopoiesis, cell proliferation and differentiation, as well as the immune system consisting of innate and adaptive immunity in the body. Interleukin-12 (IL-12) is a member of the IL-12 family of cytokines. IL-12, which is secreted by phagocytic cells in response to pathogens, is a heterodimer protein of two subunits (p35 and p40) that acts primarily during the induction of Interferonγ (IFNγ) via the signal transducer and activator of transcription 4 (STAT4) in T and natural killer (NK) cells (Guo et al., 2019). IL-12 is a cytokine that strongly induces the Th1 response. Activated macrophages by IFNγ enhance phagocytosis and bactericidal activity (Masuda et al., 2010).
It is known to be that lactic acid bacteria induce the IL-12. IL-12 activates NK cells and promotes the production of IFN-γ. IFN-γ activates macrophages, resulting in enhanced phagocytosis and bactericidal activity. We have been investigating fermented foods that activate the immune function. For that purpose, a specific antibody is required. We tried to express IL-12p35 by the usual method, but IL-12p35 was not expressed at all. In the present study, we constructed, purified human IL-12p35 and obtained a specific antibody against IL-12p35. We also purified human IFN-γ and obtained specific antibody against IFN-γ. We have established a method for expressing poorly expressed proteins. The method we have established can be applied to the purification of poorly expressed proteins and antibody production.
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