Practice of exploratory experimental teaching project based on BRET/Nanobit technology
[Objective]Based on the research transformation experiments,we introduce scientific problem-oriented exploratory experiments,lead undergraduates to establish new technological models,BRET and Nanobit,and utilize this model to explore unknown GPCR-mediated bias signaling pathways.Through teaching practice,the feasible path and practical effect of such an exploratory project in undergraduate practical teaching and innovation ability training are discussed.[Methods]Rluc8 and GFP2 proteins were used to construct fusion expression plasmids with GPCR and β-arrestin,respectively,and they were co-transfected into cells.When the GPCR is activated and β-arrestin is recruited,BRET occurs due to the close proximity of the two proteins,ensuring that the specific fluorescence signal emitted by GFP2 can be detected.At the same time,Gα(i)-GFP2,Gα(s)-GFP2,and Gα(q)-GFP2 were constructed to explore the direct interaction between GPCR and different Gα isoforms.In addition,we constructed the Nanobit test system with interested GPCR,Gα(i)-lgbit,Gα(s)-lgbit,Gα(q)-lgbit,Gβ,and Gγ-smbit.After different concentrations of ligands were added to activate the GPCR,the G protein was activated;that is,the Gα subunit was dissociated from the Gβγ subunit.Here,the luciferase activity was lost due to the separation of lgbit and smbit,and the fluorescence was reduced.The activation properties of ligands to GPCRS and the type of Gα subunit that is activated were studied by analyzing the changes in fluorescence.[Results]The BRET curves of FPR1-Rluc8 and β-arrestin1-GFP2,FPR1-Rluc8 and β-arrestin2-GFP2,were obtained,along with the EC50 value and action window of the reaction,using the BRET experiment.The results showed that FPR1 interacts with both β-arrestin1 and β-arrestin2,and the affinity with β-arrestin2 is stronger than that with β-arrestin1.This can dynamically reflect the recruitment process of β-arrestin1 and β-arrestin2 after the reaction of formyl peptide on FPR1.In addition,the activation of Gα subunits was obtained through the Nanobit experiment;for example,GPR103 acted with its ligands 26RFa and 43RFa,respectively.The Nanobit curve and the EC50 value and action window of the reaction showed that at a low concentration of 26RFa(10-9-10-8 M),GPR103 tended to couple with Gα(i)subunit,while at a higher concentration(10-8-10-5 M),GPR103 coupled with Gα(i),Gα(s)and Gα(q)subunits.At low concentration of 43RFa(<10-10 M),GPR103 was mainly coupled with Gα(q);at medium concentration(10-10-10-8 M),GPR103 tended to couple with Gα(q)and Gα(i),and at higher concentration(10-8-10-5 M),GPR103 was coupled with Gα(i),Gα(s)and Gα(q).Further,the dynamic effects of different types of Gα subunits coupled to GPCR were obtained.[Conclusions]The BRET and Nanobit systems can be used to study multiple protein-protein interactions downstream of GPCR,explore the unknown GPCR biased signal transduction,and broadly explore space in the development and application of new technologies,which is suitable for exploratory teaching experiment projects.The exploratory experimental projects can narrow the distance between specific scientific problems and undergraduates,stimulate students'internal drive to solve specific scientific problems and guide students to think deeply and innovate in practical applications.Exploratory practice teaching is expected to be an important supplement to undergraduate practice teaching and innovative ability training.
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