Study On Spycatcher As A New Label Protein

[Objective] To explore the Spycatcher can be used as a new efficient solubilization of tagged protein.[Method]1.Fusing the gene of Spycatcher and programmed death1(PD-1)and the mutation of PD-1 and FTH-PD-1 into the vector of PET21 a by the method of molecular biology.2.PET21a-Spycatcher-PD-1,PET21a-Spycatcher-MPD-1,PET21a-Spycatcher-FTH-MPD-1 were transformed into Escherichia coli.(BL21)and then inducibled expression.Then,these Escherichia coli.were ultrasonicated and the supernatant was purified by nickel column 3.Detecting the affinity of Spycatcher-MPD-1 with its ligand PD-L1 by ELISA.The experiment group and control group using Spycatcher-MPD-1 labelled with HRP and PBS respectively by coating the protein of PD-L1 into plating and then they we re all sent to be detected by microplate reader at OD450.Secondly,the protein of biotinylated PD-L1 was used to quantitative determinated the affinity to the fusion proteins by fortebio.At the same time we detected their affinity at different p H.4.PET21a-Spycatcher-PD-1 and PET21a-Spycatcher-MPD-1 labelled with fluorescence molecular of CY5.5 were used to detected the PD-L1 highly expressed on the breast cancer cell surface by fluorescence microscope and laser confocal microscope.5.We at last tried to added certain thrombin into the fusion proteins and then secendly purify the PD-L1 without Spycatcher by nickel column.[Result] 1.The sequencing result suggest that the target gene cloned into plasmid was correct.2.The purity of proteins purified by nickel column reach 85%.3.The result of ELISA suggest that OD value of experiment group is higher than the control group markedly(P<0.01).4.The result of fortebio in dicate that the affinity of PD-L1 with Spycatcher-PD-1 is about 10-8M and don't vary with the change of the pH,however,the affinity of PD-L1 with Spycatcher-MPD-1 is enhanced along with the decline of p H.5.In vitro cell research suggest that the Spycatcher-PD-1and Spycatcher-MPD-1 labelled with CY5.5 can bond to breast cancer cell surface.6.In addition,the experience of enzyme digestion of thrombin to the Spycatcher-thrombin-MPD-1 and Spycatcher-thrombin-FTH-MPD-1 indicate that the thrombin can work on the fusion proteins and attain the PD-1 and FTH-PD-1 without Spycatcher.[Conclusion] Spycatcher can be used as a new solubilization of tagged protein.