Preliminary trial—Replaceability of the Trim21-antibody interface
The corner stone of the PREDATOR Pro system is to assume that the original PRYSPRY/IgG-Fc interaction in the PREDATOR V1 and V2 system is replaceable by other protein dimerization pairs without affecting the protein degradation function (See the design page for more details).To test such hypothesis, we established a GFP targeting PREDATOR Pro system (GFP PrePro) by replacing the PRYSPRY/IgG-Fc interaction with the DocS-Coh2 modules. The constitutive dimerization of DocS and Coh2 would function as the interface to bring the truncated Trim21 into proximity with GFP protein and trigger the degradation of GFP (Fig 1A).
Figure 1. GFP PREDATOR Pro system based on the DocS-Coh2 interaction module dramatically decrease cellular GFP level throughout a long time span.(A) Schematic representation showing the design and function of GFP PrePro (B) Schematic representation showing the experimental workflow (left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (right panel). (C) Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid. (D) Representative Western blotting determining the GFP plasmid protein abundance in HEK-293T cells 48 h post co-transfected with pEGFP plasmid and GFP PrePro/control plasmid (left panel), and quantified GFP protein level in three biological replicates (right panel). Relative protein level was calculated by normalizing the gray scale data of each group to the control group. (E) Quantified fluorescent intensity in HEK-293T cell imaged 12-144 h post co-transfection with pEGFP plasmid and GFP PrePro /control plasmid (Dots with error bar), the modeling predictions were shown in curve. (F) Representative fluorescence images in (E). Fluorescent intensity of each well were calculated by averaging the fluorescent intensity in at least three random visions. Error bars represent at least three biological replicates. Statistical significance was calculated via either student t test or two way ANOVA test. ** p<0.01, *** p<0.001.
To find the optimal GFP PrePro transfection dosage, gradient amount of GFP-expression plasmid, and GFP PREDATOR Pro plasmid were co-transfected into HEK-293T cells (Fig 1B, left panel), fluorescent images were taken 48 hours post transfection. Fluorescent intensity (Click here for more about model results)was then transformed into heatmap for better visualization. Through the difference of color shade (the lighter the color, the better the degradation effect), a GFP PrePro dose dependent GFP degradation could be clearly observed. Furthermore, we provided the data to Model hoping that they could obtain the optimal concentration ratio of GFP and GFP PrePro plasmids.(Click here to know more about model)With their advice, the plasmid ratio was set as 1:1 in further experiment.
Under such transfection ratio, further experiments were conducted in HEK-293T cells to characterize the performance of the GFP PrePro system. Similar to previous ratio screening results, ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection comparing to the pEGFP plasmid and control plasmid co-transfected group(Fig 1C). Subsequently, Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group with the appearance of GFP PrePro (Fig 1D), which confirmed that PREDATOR Pro could be used to degrade target protein with high efficiency.
To further characterize the durability of GFP degradation by GFP PrePro, we imaged cells at 12, 24, 36, 48, 72, 96, 108 and 144 hours after transfection. It was surprising to observe that significant lower GFP fluorescence could be observed in GFP PrePro expressing group comparing to the control group in every time point from 12 hours to 144 hours post transfection (Fig 1E and F). Of note, such trend was also well aligned to the prediction of our mathematical model (Fig 1E, shown in curve).
After consulting Professor Zanxian Xia (Click here for more about Interview) and presenting our data in CCiC meetup. We were suggested to change the GFP reporter into a Dual Luciferase Reporter system (Fig 2A) to normalize irrelevant factors affecting GFP abundance (See design page for further information about the designing of this reporter). With this new reporter, the abovementioned experiments were repeated and the results clearly showed that GFP PrePro could degrade the target protein significantly (Fig 2B).
Figure 2. Dual luciferase assay showing the GFP degradation mediated by GFP PrePro system.(A) Schematic representation of the dual luciferase system indicating the cellular GFP level. (B) Dual luciferase assay of HEK-293T cells 48 h post co-transfection with pEFR plasmid and GFP PrePro/control plasmid. pEFR indicates “plasmid expressing EGFP-FLuc-2A-Rluc”. Error bars represent at least three biological replicates. Statistical significance was calculated via student t test. *** p<0.001.
These results implied that the PREDATOR Pro system, in which the original PRYSPRY/IgG-Fc interface in the PREDATOR V1 and V2 system were replaced into other protein dimerization pairs, could still efficiently degrade target protein. This design then provided new interface to rewire the signal-inducible protein-protein interaction to the target protein degradation process.
Towards signal responsiveness:
To push our new PREDATOR Pro system towards signal responsiveness, we further engineered a Rapamycin-induced PREDATOR Pro system (RiPrePro-1 or RiPrePro1.0) by replacing DocS-Coh2 with FRB-FKBP, which enable rapamycin-induced dimerization and subsequent formation of ternary degradation complex, leading to the degradation of target protein (Fig 3A). To test whether the GFP levels can be tuned and continuously regulated by rapamycin, HEK-293T cells were co-transfected with GFP expressing plasmid and RiPrePro1.0 plasmid/empty vector. Fluorescent imaging showed slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group comparing to the control group (Fig 3B and 3C) under 1 ng/μL rapamycin induction.
Figure 3. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.(A)Schematic representation showing the design of Rapamycin induced PREDATOR Pro (RiPrePro-1) and its degradation mechanism (B) Representative fluorescence images of the RiPrePro-1 transfected group and negative control group transfected with an empty vector. HEK293T cells in both groups were co-transfected with GFP-expression plasmid. Cells were induced with 1 ng/μL rapamycin 24 h post transfection. Images were taken 48 h post induction. (C) Quantification of fluorescent intensity in (B) and its parallel biological replicates. Statistical significance was calculated via student t test . *** p<0.001.
To improve the degradation effect of our RiPrePro system, we approached our modeling group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis among all parameters in our model (Click here to know more about Model), we noticed that the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pair (Fig 4). In the case of RiPrePro system, such result implied that the degradation effect can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part of the protein.
Figure 4.Sensitivity Analysis of related parameters regarding the protein interaction intensity.
As expected, the degradation effect could be significantly improved with the increasing concentration of rapamycin (Fig 5). However, it has been well established that exorbitant amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiplying FKBP copies for a higher intensity.
Figure 5. Heatmap of fluorescence data detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin.
From single copy to double copy:
significantly enhanced degradation effect
With the implications obtained from the model, we changed the GFPnano-FKBP unit in the RiPrePro1.0 into GFPnano-FKBP-FKBP unit to further explored whether our Rapamycin-induced PREDATOR Pro system with two FKBP fragment (RiPrePro-2 or RiPrePro2.0) could show better performance. It was observed that under 2 ng/μL rapamycin induction, the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group transfected with pEGFP and empty vector (Fig 6A). A significantly improved GFP degradation (shown by Fluc/Rluc ratio) was also observed in RiPrePro2.0 expressing group comparing to RiPrePro1.0 expressing group and control group in 12, 24, 48 and 72 hours post 2 ng/μL rapamycin induction (Fig 6B).
Then, further characterization was performed on RiPrePro2.0 system. Upon gradient amount of rapamycin stimulation, RiPrePro2.0 showed dose dependent GFP degradation ability in response of rapamycin concentration (Fig 6C). Western Blotting also showed ~40% decrease on GFP abundance in HEK-293T cells expressing RiPrePro2.0 comparing to the control group, under 2 ng/μL rapamycin induction for 48 h (Fig 6D), which demonstrated that RiPrePro2.0 could degrade target protein with high efficiency under the control of rapamycin. Finally, to test whether RiPrePro2.0 works in different host cells, we co-transfected RiPrePro2.0 plasmid with pEFR plasmid into different cell lines. As shown in Fig 6E, significant lower Fluc/Rluc ratio could be observed in RiPrePro2.0 group comparing to the control group among all test cell lines under 2 ng/μL rapamycin induction. This result further demonstrated the robustness of the RiPrePro2.0 system.
Figure 6. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module. (A) Representative fluorescence images of HEK-293T cells carrying pEFR and RiPrePro-2 plasmids in comparison to the cells carrying pEFR and empty vectors 48 h post 2 ng/μL rapamycin induction. (B) Dual luciferase assay showing the normalized GFP abundance in pEFR transfected HEK-293T cells co-transfected with RiPrePro-2 or empty vector. Cells were harvested 24, 48, or 72 h post 2 ng/μL rapamycin induction. (C) Dual luciferase assay showing the normalized GFP abundance in HEK-293T cells co-transfected with pEFR and RiPrePro-2. Cells were treated with respective amount for rapamycin for 48 hours before harvested. (D) Western blotting determining the GFP protein abundance in pEFR transfected HEK-293T cells co-transfected with RiPrePro-2 or empty vector. (E) Dual luciferase assay showing the normalized GFP abundance in different host cells co-transfected with pEFR and RiPrePro-2/empty vector. Error bars represent at least three biological replicates, Statistical significance was calculated via student t test. * p<0.05, ** p<0.01, *** p<0.001.
In general, these results showed that our PREDATOR Pro system can be successfully engineered to degrade the target protein under the control of exogenous signals with decent performance and satisfying robustness. The engineering success of these designs also showed the potential of PREDATOR Pro system to be further developed into a novel synthetic biology toolbox for controlled protein degradation.