Team:NUDT CHINA/Results

Designed Protein Degradation Method Based on

Trim21 And Nanobody              -- Results

Proof of concept：

Figure 1. Degradation of GFP by GFP PREDATOR. (A) Schematic representation showing the GFP degradationmechanism by GFP PREDATOR. (B) Immuno precipitation assay verifying the binding of GFP-nanobody-Fc and GFP. (C) Fluorescence images indicated a significantly decrease in GFP fluorescence in GFP-PREDATOR transfected group.(D) Western blotting determining the expression level of GFP-nanobody(nano)-IgG Fc(IgG Fc), GFP, HA Tag-Trim21.(E and F) Western blotting assay showing the degradation of GFP in GFP PREDATOR transfected groups in different time after transfection.

To verify the degradation efficiency of target protein, we first set up a proof of concept experiment in mammalian cell culture with GFP, a well-established green fluorescent protein used in cell biology research,as the target protein.

For such matters, we first constructed the GFP PREDATOR plasmid expressing following parts: 1) the GFP-nanobody (nano)-IgG Fc (IgG Fc), encoding the fusion protein of GFP single domain antibody (nanobody, or nano) and human IgG Fc fragment to target and bind exogenously expressed GFP in cytoplasm, forming the nanobody-GFP complex; 2) HA Tag-Trim21, which recognizes the Fc fragment of the nanobody-GFP complex and induce the ubiquitination of complex, subsequently leading to the proteasomes-dependent protein degradation. HA Tag was added to N-terminal of Trim21, making it easier to be detected by western blot assay. The plasmid which did not express nanobody-Fc and Trim21 was set as the control of PREDATOR .

To experimentally proved the effectiveness of PREDATOR, immune precipitation was performed to validate the binding of GFP nanobody and GFP. Western blotting results showed a significant co-precipitation of GFP nanobody and GFP protein (Figure 1b), suggesting a strong interaction of GFP nanobody and GFP protein in HEKK293T cells. To test whether GFP PREDATOR system could induce the degradation of GFP, GFP-expression plasmid and GFP PREDATOR plasmid was co-transfected to HEK293T cells to express GFP and introduce GFP PREDATOR system to cells. A significant decrease of green fluorescence was observed in GFP PREDATOR transfected groups 48 hours after transfection indicating the decrease of GFP protein level (Figure 1C). Subsequently, Western blotting analysis was performed to examine the level of the key components of GFP PREDATOR: the target protein---GFP, GFP-nanobody (nano)-IgG Fc(IgG Fc)--- the bridge to connect the target and ubiquitination reaction, and E3 ligase Trim21. As shown in the results, GFP was significantly degraded to about 30% of original level with the appearance of GFP-nanoand HA-Trim, which confirmed that PREDATOR could be used to degrade target protein with high efficiency.

To further reveal the durability of GFP degradation by GFP PREDATOR , we collected cells of different time after transfection, including 12, 24, 36, 60, 72hours. Then, western blotting assay was performed to quantify the level of GFP protein in these cells to track the expression level of GFP. As is shown in Figure 1E, the GFP level of control group (transfected with GFP-expressing plasmid and PREDATOR control) increased in 12~60 hours, indicating the expression of GFP by GFP-expression plasmid. While the GFP level in cells transfected with PREDATOR was significantly lower than control in each time point. Of note, the degradation efficiency remain increasing even 72h after transfection (53.50% in 60h and 62.38% in 72h), indicating the durability of our PR PREDATOR.

Demostration：

Figure 1. Degradation of GFP by GFP PREDATOR. (A) Schematic representation showing the GFP degradationmechanism by GFP PREDATOR. (B) Immuno precipitation assay verifying the binding of GFP-nanobody-Fc and GFP. (C) Fluorescence images indicated a significantly decrease in GFP fluorescence in GFP-PREDATOR transfected group.(D) Western blotting determining the expression level of GFP-nanobody(nano)-IgG Fc(IgG Fc), GFP, HA Tag-Trim21.(E and F) Western blotting assay showing the degradation of GFP in GFP PREDATOR transfected groups in different time after transfection.

Utilizing PR PREDATOR to determine the effect of ErbB3 in the proliferation regulation of human breast cancer MCF7 cells

Here, we demonstrated how PR PREDATOR can be used in protein researches as a promising substitute of current siRNA inhibitors by downregulating and establish its regulatory relationship with proliferation.

Utilizing PR PREDATOR to determine the effect of ErbB3 in the proliferation regulation of human breast cancer MCF7 cells

We first collected different kinds of human cancer cell lines including A549 (lung adenocarcinoma), MCF7 (breast cancer), HepG2 (HCC), Hela and HEK293T to detect ErbB3 protein expression. Results show that ErbB3 has the highest expression in MCF7 (Figure 2A). Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells and inhibits proliferation of MCF7 cells has been reported in the literature. So, we introduced ErbB3 PREDATOR system into MCF7 cells, ErbB3 expression was significantly decreased by westernblotting (Figure2B) and inhibits proliferation of MCF7 cells by CCK8-assay(Figure 2C).