Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

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Immune regulation is a complex biological signaling pathway in which several classes of biomolecules and small molecules play a complacent role to mediate this process. Glycoimmunology is a rapidly evolving research area that deals with the structure, binding interactions and immunological functions of glycans. Great deal of information regarding proteins and nucleic acids in molecular recognition events have been established owing to their well-established structural features and straight forward replication, transcription and translation principles. However considering the complexities of template free synthesis and structural heterogeneity, role of carbohydrates in immune regulation are still unsung to a large extent. In the current review, we illuminate the canonical structural features, emerging and significant pathophysiological functions of glycosaminoglycans (GAGs), the negatively charged linear carbohydrate molecules that are primarily present on all types of cell surfaces and extra cellular matrix. A snap shot of their association with protein counterparts of diversified protein families has been updated exclusively to provide mechanistic insights into their cellular signaling functions. Eventually, this review throws light on the recent biomedical/biotechnological advances of GAG based biomarkers, nutraceuticals, therapeutics, and nanocomposites for inflammatory, immune disorders and their invaluable contribution in tissue engineering.

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