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CONTRIBUTION

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New experimental data for previous parts

ErbB3-composite

This part is a recombinant fusion protein consist of the single-chain antibody of ErbB3 (ErbB3 ScFv) and Fc fragment of human IgG (hIgG1-Fc). The part, submitted in 2018 by team NUDT_CHINA, recognized target protein ErbB3, and trigger the Predator-mediated degradation of ErbB3

However, the in vitro functional characterization of this part is lacking.

To validate the protein degradation efficiency of the ScFv-based ErbB3 Predator system, we transfected MCF7 cells with plasmids expressing ErbB3 ScFv-linker-Fc fusion protein and HA- Trim21. Western Blotting analysis showed that with the high expression of ErbB3 ScFv-linker- Fc fusion protein and HA-Trim21, the ErbB3 protein level decreased dramatically in cells transfected with ErbB3 Predator (approximately 30%, Fig 1A and B). This result demonstrated that the ErbB3 ScFv-linker-IgG Fc could successfully degrade the target membrane protein ErbB3. (Click BBa_K2653006 for experiment data.)

Figure 1. Western blotting assay showing the components of ErbB3 Predator and the degradation of ErbB3.

hsaGlucagon ->GGGGS ->hsaIgG-Fc ->P2A ->HA ->hsaTrim21

This part is used to degrade endogenous glucagon receptor (GCGR). However, proper control groups were still needed to prove its degradation capacity and understand the mechanism underlying the degradation processes. Therefore, two core parts in the GCGR Predator, hsaGlucagon-IgG Fc and hsa-Trim21, were introduced into cells separately, and western blotting was performed to further prove that the GCGR predator is capable of triggering GCGR degradation.

Results indicated that GCGR Pre. transfected cells showed a ~40% lower GCGR level compared to the vector control (Fig 2A and B). Besides, there is no significant difference between the control group and groups that only overexpressed Glucagon-linker-IgG Fc (Gcg-Fc) or Trim21, indicating individual components in GCGR Predator cannot trigger effective degradation of GCGR. Also, we found that both in four groups, Glycosylated GCGR (Glu-GCGR, ~64kDa) showed no difference in expression level. These results demonstrated that both Trim21 and Gcg-Fc are needed to achieve effective degradation of target protein GCGR.(Click BBa_K3064014 for experiment data.)

Figure 2.(A) Western blotting assay showing the degradation of GCGR and Glu-GCGR in the GCGR Predator transfected group and its quantified results (B).

New parts

DocS

The DocS¹ module comes from C. thermocellum and it could recognize and bind tightly to complementary Coh2 modules. The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions. (Click BBa_K3396000 for experiment data.)

Coh2

The Coh2¹ module comes from C. thermocellum and it could recognize and bind tightly to complementary DocS modules harbored by each of the catalytic subunits. The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions.(Click BBa_K3396001 for experiment data.)

1 Barak, Y. et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction. Journal of Molecular Recognition 18, 491-501, doi:10.1002/jmr.749 (2005)

Team:NUDT CHINA/Contribution