In our iGEM 2020 project, tremendous amount of engineering effort has been made to bring the signal responsive feature into the PREDATOR system we kept working on for years (Figure 1). As the most important foundation of the PREDATOR Pro system, the successful reengineer of the Trim21 protein is considered as the most important achievement we made this year. Here in this Engineering Success page, we will discuss how Trim21 protein was engineered to reach our needs and demonstrate the functional validation data of our new part BBa_K3396009, also termed as GFP PrePro, showing that it worked as we expected.
Brain storming and literature research
In 2018 and 2019, we have demonstrated the PREDATOR system, an effective endogenous protein degradation system that allows for direct regulation of target protein degradation. Although we have demonstrated that the PREDATOR system can be controlled by different signal responding promoters, thus provided a controllable protein degradation method, we also noticed that such transcriptionally-controlled feature maintains improvable to further decrease the response time lag and improve robustness.
Achieving direct signal responsiveness requires a proper interface to rewire the signal induced protein-protein interaction or other biochemical reactions into the PREDATOR system. Hence, we reanalyzed our previous working model. In the previous PREDATOR system, the nanobody-IgG Fc fusion protein links target protein and Trim21 protein via two important protein-protein interactions. Among them, the nanobody-target interaction determines the specificity of the system, thus remains untouchable. On the other hand, the second key interaction occurs between the IgG Fc domain and the Trim21 protein, seems replaceable. Further analysis on the structure and working mechanism of Trim21 also showed that Trim21 interacts with IgG Fc domain via PRYSPRY domain located on its carboxy terminus.
Modularization and Design
Building on our literature research, we determined to try replacing the Trim21 PRYSPRY domain and the IgG Fc domain linked with the nanobody into other protein dimerization pairs, either constitutive or signal-regulated, to create an interface that direct controls of the protein degradation. Since the nanobody functions as the targeting module, we then replaced the IgG Fc domain into Coh2, a protein that interacts with DocS constitutively. Also, we truncated the Trim21 protein by removing the PRYSPRY containing 268-475 aa and termed the remining 1-268 aa of Trim21 the functional module. The functional module was then fused with a DocS protein on its C terminus to interact with the Coh2 fused GFP nanobody to achieve GFP targeting.