The Large-scale Expression Of GPCRs

The family of G protein coupled receptor (GPCRs) are the most signal transduction proteins in membrance proteins. Their signal molecules include photon, odorant, ion, lipid, hormone, neurotransmitter, metabolin, and so on. And they play important roles in a lot of physiological processes, for example, contraction and relaxation of muscle, signal transmission in central nervous system, convene of immune cells, function of vision, function of gustation, cell proliferation and differentiation. So now GPCRs are regarded as the best targets of drugs to cure relative diseases. But the very low expression of functional GPCRs seriously limit many research techniques, as nuclear magnetic resonance (NMR), X-ray. The development of technologies using for GPCRs’expression and the increasing of ratio for functional GPCRs have been hot spot in the area of structural biology.In several proteins’expression systems, the mammal cells are ideal systems to express GPCRs. According to requirements of NMR, this paper has optimized many conditions, such as concentration of Ca2+, composition of culture medium. We have achieved suspension culture of mammal cell HEK293S GnTI" in large-scale, and the density of cell growth in suspension has been increased about30%. Along with screening stable cell strain which can yield Opsin protein at high levels and has the Tet system, we greatly increased the level of Opsin output in the same criteria. Also, we are screening stable cells expressing another two GPCRs (CX3CR1, ADRB3). The results of these research will achieve large-scale expression of Opsin, CX3CR1, ADRB3, which would be important progress to study relative diseases. And the expressed GPCRs can satisfy demands of different research techniques, such as X-ray crystallography.