Difference between revisions of "Team:NUDT CHINA/Results"

Proof of concept：

Figure 1. Degradation of GFP by GFP PREDATOR. (A) Schematic representation showing the GFP degradation mechanism by GFP PREDATOR. (B) HEK293T cells were transfected with GFP PREDATOR plasmid and GFP-expressing plasmid, fluorescence images indicated a significantly decrease in GFP fluorescence. (C). ? (D)Western blotting determining the expression level of GFP-nanobody(nano)-IgG Fc(IgG Fc), GFP, HA Tag-Trim21.

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. (好处加一句?)

We construct the GFP PREDATOR plasmid expressing following parts :1) the GFP-nanobody(nano)-IgG Fc(IgG Fc), which expressing the infusion protein of GFP single domain antibody (nanobody, or nano) and human IgG Fc fragment to target and bind to free 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. immunoprecipitation was performed to validate the binding of GFP nanobody and GFP. Results showed the complexes precipitated with ProteinA/G by Fc fragment which directly linked to GFP nanobody contained GFP protein (detected by Western Blotting), this verified the binding between GFP and GFP nanobody. 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 ectopically express GFP and introduce GFP PREDATOR system to cells. 48 hours after transfection, we found the green fluorescence was significantly decreased comparing with control, indicating the decrease of GFP protein level (Figure 1C). Subsequently, Western blot 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-nano and 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 measure the degradation level of GFP. As is shown in Figure 1E, the control group (transfected with GFP-expressing plasmid and PREDATOR control) was increasing in 12~60 hours, indicating the expression of GFP by GFP-expression plasmid. While the GFP level in cells transfected with PREDATOR was less than control in the same transfection time. 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.