Team:NUDT CHINA/Proof Of Concept

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PROOF OF CONCEPT

Whether it works

Overview

Regulating protein abundance is the basis of constructing complicated synthetic regulatory networks. Seeing tremendous achievements made recently on genomic, transcription or translation level to regulate protein synthesis¹. We noticed that tools regulating the protein degradation have been seriously outnumbered in the synthetic toolbox. Hence, in order to expand our tools to regulate protein abundance, we hope to develop a targeted protein degradation system directly controlled by exogenous signal, termed PREDATOR Pro system, on the basis of the PREDATOR system we reported in iGEM 2018 and 2019.

By looking into the working mechanism of PREDATOR system, we hypothesized that the PRYSPRY domain of the Trim21 can also be replaced with other protein domains that can dimerize with other proteins under given signal input. This new protein dimerization pair could then provide a new interface for exogenous signal control (see the Design page for more details)

Figure 1. Development process of our iGEM2020 project

The development of the whole project is shown in Figure 1. Specifically, to validate our hypothesis and prove our basic concept, we replaced the original [antibody Fc domain – Trim21 PRYSPRY domain] interface with the DocS-Coh2 dimerization pair. The constitutive dimerizing feature of DocS and Coh2 would help us to understand whether the [antibody Fc domain – Trim21 PRYSPRY domain] interface can be replaced without affecting the protein degradation function of the remaining Trim21 truncated protein. Subsequently, we then engineered a rapamycin-induced PREDATOR Pro system to demonstrate that our system can work for our purposed implementation.

Here we mainly show the data causally related to the experiments mentioned above under optimized parameter and plasmid design. Other supporting data and how our modeling results and HP feedbacks affect the development of our project are located in the Result page (  click the link here to go to the Result page)

Replacing the Trim21-antibody interface

The corner stone of the PREDATOR Pro system is to assume that the original PRYSPRY/IgG-Fc interface (Fig 2A, left panel) in the PREDATOR V1 and V2 system (see our iGEM 2018 and 2019 iGEMprojects for details) is replaceable by other protein dimerization pairs without affecting the protein degradation function. To test the replaceability of the interface, we replaced the natural PRSPRY-Fc pair in the GFP targeting PREDATOR system with DocS-Coh2, a previously reported constitutive dimerization pair (Fig 2A, right panel). The new system was termed as ‘GFP PrePro’.

Figure 2. Characterization of GFP PREDATOR Pro system based on the DocS-Coh2 interaction.(A) Schematic representation showing the design and function of GFP PrePro. (B) Representative Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid. (C) 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. (D) 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. (E) 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<0.01, *** p<0.001.

    To validate the function of GFP PrePro, HEK-293T cells were co-transfected with GFP expressing reporter plasmids and GFP PrePro expressing plasmids as the experimental group (GFP PrePro group). Florescent imaging showed that comparing to the control group in which HEK-293T cells were co-transfected with GFP expressing reporter plasmids and empty vector (Ctr group), GFP fluorescence in GFP PrePro group was ~60% lower than the Ctr group. Western blotting analysis also showed ~60% lower GFP abundance in GFP PrePro group (Figure 2B). Fluorescent imaging throughout the 144 hours post transfection also showed significantly lower GFP florescence in GFP PrePro group comparing to the Ctr group (Figure 2C, shown in dots with error bar, p<0.001, tested with two-way ANOVA analysis). Such result was in alignment with the modeling results, which predicted a significantly lower fluorescent intensity in GFP PrePro group (Figure 2C, shown in curve). Moreover, we used a dual luciferase reporter (pEFR), in which firefly luciferase (Fluc) was fused with GFP, to obtain more accurate GFP abundance changes. Fluc activity was used as an indicator of GFP abundance, and the activity of a constitutively expressed Renilla luciferase (Rluc) reporter was used to normalize the noise caused by irrelevant factors such as transfection efficiency, cellular protein synthesis and cell growth. Results also showed significant lower (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group. These results suggested that the original PRYSPRY/IgG-Fc interface in the PREDATOR system could be replaced with other protein dimerization pairs while maintaining the protein targeting and degradation activity.

Towards Signal Responsiveness

To further push the PREDATOR Pro system towards direct responsiveness on exogenous signals, we then constructed Rapamycin Induced PREDATOR Pro (RiPrePro) system by replacing the DocS-Coh2 dimerization pair in the GFP PrePro into well-characterized, rapamycin inducible FRB-FKBP dimerization pair (RiPrePro1.0 or RiPrePro-1, Fig 3A). With the advices from the modeling group, RiPrePro system with two FKBP copies fused with GFP nanobody was also constructed (RiPrePro2.0 or RiPrePro-2, Fig 3A) to improve the system performance (for detailed information on the modeling results and feedbacks, click here to go to the Result page). Also, dual luciferase reporter pEFR was used to normalize the difference on protein synthesis caused by rapamycin.

Figure 3. Characterization of Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.(A) schematic representation of the protein design of the RiPrePro1.0 and 2.0 systems. (B) Dual luciferase assay showing the normalized GFP abundance in pEFR transfected HEK-293T cells co-transfected with RiPrePro-1/-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.

Similarly, for RiPrePro1.0/2.0 group, HEK-293T cells were co-transfected with pEFR and plasmid expressing RiPrePro1.0/2.0. In Ctr group, HEK-293T cells were co-transfected with pEFR and empty vector. Dual luciferase assay showed that both RiPrePro1.0 and RiPrePro2.0 groups showed significantly lower Fluc/Rluc ratio comparing to the Ctr group at 24, 48 and 72 hours post 2 ng/μL rapamycin induction. The normalized GFP abondance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig 3B), indicating an improved degradation efficiency under increased FKBP copies. Further analysis on RiPrePro2.0 further revealed that the GFP degradation activity of RiPrePro2.0 was dose dependent to the rapamycin concentration (Fig 3C). Similarly, western blotting analysis showed ~40% lower GFP protein level in RiPrePro2.0 group comparing to the Ctr group (Fig 3D), which is in alignment with the dual luciferase assay results. Moreover, 30%-60% reduction on Fluc/Rluc ratio in the RiPrePro2.0 group comparing to the Ctr group could be observed in different host cell lines we tested 48 h post 2 ng/μL rapamycin induction, suggesting good robustness of such RiPrePro system (Fig 3E).

Above all, we not only proved the basic concept of our protein design, but also demonstrated rapamycin responsive protein degradation using PREDATOR Pro system. These results suggested that our system could work for our purposed implementation.

Reference

1   Xie, M., Haellman, V. & Fussenegger, M. Synthetic biology—application-oriented cell engineering. Current Opinion in Biotechnology 40, 139-148, doi:10.1016/j.copbio.2016.04.005 (2016).

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