Orphan G Protein Coupled Receptor Library And The Optimal Expression Of Human Interleukin-6

G protein-coupled receptors(GPCRs) are the largest group of the cell membrane receptors playing critical biological functions. Human genome encodes approximately 400 non-olfactory GPCRs that are thought to have endogenous ligands. So far, the endogenous ligands of approximately 120 GPCRs are remained unknown and these receptors are termed as orphan GPCRs. Protein/peptide hormones can activate about 50% of GPCRs with known endogenous ligands, being the most important group of the endogenous GPCR ligands. Thus, we deduced that secretory proteins and peptides are important candidates of orphan GPCR ligands. To identify the protein/peptide ligands, we generated two main orphan GPCR libraries that over-express 95 cloned orphan human GPCRs in mammalian cells. In one library, the orphan human GPCRs were cloned into pc DNA vector and the tagless GPCRs were constitutively over-expressed in the host mammalian cells(such as HEK293 T cells). Through co-expression of a Nano Luc reporter controlled by downstream GPCR signaling pathways, the protein/peptide ligands can be screened by the sensitive reporter assay. In the other library, the orphan human GPCRs were cloned into a p TRE3 G vector and the C-terminally EGFP-tagged GPCRs were over-expressed in the host mammalian cells under a controlled manner. Using the fluorescent probe-labeled proteins and peptides, the GPCR ligands can be screened under fluorescent microscopy based on the ligand-induced GPCR internalization. Our orphan GPCR libraries will facilitate the screening of the protein/peptide ligands of these orphan GPCRs that are potential therapeutic targets.In another work, we improved the production of biologically active Interleukin-6(IL-6) in E. coli using Nanoluciferase(Nano Luc) reporter and detected the role of IL-6 in STAT3 signaling pathways in HEK293 T cells by processing bioluminescent quantitative assays. Nanoluciferase(Nano Luc) is the so far brightest bioluminescent reporter developed by Promega in 2012. Furthermore, we fused Nano Luc to human leukemia inhibitory factor(LIF) for both efficient overexpression and various bioluminescent quantitative assays. Since biologically active IL-6 plays significant roles in numerous diseases and in body system, the more biologically active IL-6 is required for research. In previous work, the final yield of biologically active IL-6 is still low. In the present work, we fused Nano Luc to IL-6 as a novel quantitative protein fusion tag to improve the final yield of biologically active IL-6 and for bioluminescent quantitative assays.