Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 英文参考文献.docVIP

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Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 英文参考文献.doc

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Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 英文参考文献

ProductionofaBioengineeredG-ProteinCoupled ReceptorofHumanFormylPeptideReceptor3 XiaoqiangWang2,ShuguangZhang1* 1LaboratoryforMolecularFabrication,CenterforBitsandAtoms,MassachusettsInstituteofTechnology,Cambridge,Massachusetts,UnitedStatesofAmerica,2Center forBioengineeringandBiotechnology,ChinaUniversityofPetroleum(EastChina),Qingdao,Shandong,China Abstract G-proteincoupledreceptors(GPCRs)participateinawiderangeofvitalregulationsofourphysiologicalactions.Theyare also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdlesneedtobeovercome;theyincludeoverexpression,solubilization,andpurificationoflargequantitiesoffunctional andstablereceptorsonaregularbasis.Herewereportmilligramproductionofahumanformylpeptidereceptor3(FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineeredFPR3wasoverexpressedinstabletetracycline-induciblemammaliancelllines(HEK293S).Afterasystematic detergentscreening,fos-choline-14(FC-14)wasselectedforsubsequentsolubilizationandpurificationprocesses.Atwo- steppurificationmethod,immunoaffinityusinganti-rho-tagmonoclonalantibody1D4andgelfiltration,wasusedtopurify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptorwascorrectlyfoldedwith.50%a-helix,whichissimilartootherknownGPCRsecondarystructures.Ourmethod canreadilyproducemilligramquantitiesofhumanFPR3,whichwouldfacilitateindevelopinghumanFPRastherapeutic drugtargets. Citation:WangX,ZhangS(2011)ProductionofaBioengineeredG-ProteinCoupledReceptorofHumanFor