engleski

Production and high-throughput site-directed mutagenesis of human cannabinoid CB2 receptor

Human cannabinoid receptor type2 (CB2) is a class A G protein-coupled receptor (GPCR). It is a part of our endocannabinoid system and a potential target for treating many diseases. Therefore, much effort is put into elucidation of structure and function of that valuable receptor. Within this thesis, CB2 is expressed in human cells, both transiently and stably, and its expression verified by confocal microscopy. Effect of various detergent and cannabinoid ligand present in buffers used for receptor solubilisation was evaluated. In addition, CB2 is characterised by fluorescence size-exclusion chromatograpy (FSEC). Furthermore, constructs fused with SNAP-tag were used for establishing a homogeneous time resolved fluorescence (HTRF) assay that would allow for high-throughput thermostability screening of hundreds of CB2 variants. For that purpose, CB2 mutants were produced by two- fragment PCR mutagenesis. Mutations were designed both rationally, based on thermostabilisation of other class A GPCRs, and by systematic alanine scanning mutagenesis. However, the HTRF assay still has to be optimised for their analysis.

CB2 ; GPCR ; conformational stabilisation ; HTRF ; high-throughput PCR mutagenesis ; alanine scanning mutagenesis

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