Study On Detection Method Of N-Glycosylase Activity Based On Red Fluorescent Protein Dimer

Red Fluorescent Protein(RFP)is a light-emitting protein that allows you to observe cell activity,perform in-depth protein research.The red fluorescent protein heterodimer is formed by the polymerization of two monomeric proteins ddRFPA1 and ddRFPBl.The monomer is derived from the directed evolution of dTomato gene.The chemical properties of the two proteins are significantly different.When they are in the monomeric state,ddRFPAl exhibits weak fluorescence,ddRFPB1 has no fluorescence,and it forms a bright red dimer upon binding,which results from the establishment of a protein-protein that spontaneously mediates fluorescence between the two monomers interface.If N-glycosylation is introduced on the two monomeric proteins,the interface of the monomer can be changed to prevent the two from binding to each other,so that the red dimer can be restored to the monomer state,and the fluorescence is significantly reduced.PNGase is one of the most effective enzymes for deglycosylation,which deglycosylates two ddRFP monomers and restores the protein-protein interface to form a highly fluorescent dimeric state.Therefore,the activity of the PNGase enzyme can be detected by varying the fluorescence intensity of the RFP.In this study,site-directed mutagenesis was used to introduce N-glycosylation sites into ddRFP.Red fluorescent dimers were obtained after the mutants were co-expressed.A yeast system was established to modify the proteins to obtain glycosylated protein products;the target protein was mixed in vitro.In the experiment,the activity of PNGase was detected by measuring the fluorescence intensity change after deglycosylation of the mixture.The specific research and results are as follows:1.Site-directed mutagenesis and mutant co-expression of ddRFPAl,ddRFPB1Based on the gene sequences of ddRFPA1 and ddRFPB1,primers containing mutation sites were designed.The mutation sites were Lys 139A,Tyr 146A,Phe 178A,Val196A,Glu160B,Val196B mutations were Asn,and Val176A and Glu101B mutations were Ser.At last,The RFP139A,RFP146A,RFP176A,RFP178A,RFP196A,and RFP196B mutants were successfully obtained,and mutations in RFP101B and RFP160B failed.Wild-type ddRFPA1 and mutant A(all mutants of ddRFPAl)were transformed into BL21 competent cells containing the ddRFPB1 and RFP196B genes respectively.Expression results showed that ddRFPA1/ddRFPB1,ddRFPA1/ddRFP196B,ddRFP139A/ddRFP196B were formed in BL21 cells and all three dimers could emit bright red fluorescence,which was significantly different from the ddRFPA1,ddRFPB1,and RFP196B in the control group.2.Eukaryotic and yeast expression of ddRFPAl,ddRFPBl and mutantsddRFPA1,ddRFPB1 and their mutants were designed by primers,amplified by PCR,ligated into intermediate vector,digested and then ligated into eukaryotic vector pPICZαB-H.The 5AOX primers were used for sequencing.All the genes were successfully cloned.GS115 yeast competent cells were prepared by lithium acetate method,and the recombinant plasmid of the fluorescent protein gene and pPICZaB-H vector was linearized by MSSI,then transformed into yeast cells to obtain a successful transformant,and the transformants were expanded and cultured one week after expression culture.The target protein expression supernatant was obtained and verified by western blot.The results showed that ddRFPAl and ddRFPBl and their mutants could be successfully expressed in the yeast system,and the expressed protein was the same size as the target protein band.3.The application of red fluorescent dimer in the detection of PNGase activityThe second chapter of the yeast expression supernatant was ultrafiltrated,and 100 ul of ddRFPA1 and ddRFP196B were respectively mixed at a ratio of 1:1 in a 96-well microtiter plate.100 ul of each combination of ddRFP139A and ddRFP196B was mixed 1:1 and PNGase was added.Enzymes were incubated at 37℃ for 16 h in a fluorescence plate reader and their fluorescence values(ex/em:535 nm/605 nm)were measured every 2 h.The results showed that the combination of ddRFPA1/ddRFP196B showed a significant increase in the fluorescence value under the action of PNGase,which was significantly different from the blank control group.There was no significant change in the fluorescence values of the combination ddRFP139A/ddRFP196B.This study provides structural assumptions for the study of fluorescent protein dimers.Validation shows that protein glycosylation affects the structure of the dimer and thus affecting its fluorescence intensity.This provides a new direction for the study of fluorescent proteins;PNGase activity assay generally uses chemical methods.After the substrate is digested with PNGase,the released N-glycans are labeled with the fluorophore 2-aminobenzamide(2-AB)and detected with high performance liquid chromatography(HPLC-FD).this article is a biological method for detecting PNGase enzyme activity by fluorescence intensity,and has a good application prospect in substrate detection with high safety and convenience requirements.