Difference between revisions of "Part:BBa K4839024"

Revision as of 12:35, 12 October 2023

pGK-GFP-IRF4

This part is design for the validification of our BioPROTC system. In our design, we fused GFP and IRF4 together and thus we can use both anti-GFP or anti-IRF4 BioPROTAC to detect the degradation efficiency of both protein.

We would like to construct GFP-IRF4 using visualization methods to simulate the degradation process of IRF4 by anti-IRF4 BioPROTAC. Therefore, we have proposed the following design, and we have also designed alternative options, namely pcDNA3.1-CMV-FLAG-IRF4 and pcDNA3.1-CMV-HA-IRF4. The sequence diagrams of GFP-IRF4 and the alternative options FLAG-IRF4 and HA-IRF4 are shown in Figure 1.

Figure 1. The sequence diagrams of GFP-IRF4 and the alternative options FLAG-IRF4 and HA-IRF4.

Since we were unable to complete the construction of GFP-IRF4 within a short period of time, we have chosen to use pcDNA3.1-CMV-FLAG-IRF4 and pcDNA3.1-CMV-HA-IRF4 as our targets for degrading IRF4. We have successfully achieved expression and conducted the following validations of the functionality of anti-IRF4 BioPROTAC.

we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 3-2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.

Figure 2. Western blot signing degrdation of IRF4. "D" represents doxycycline.

Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.

[1] Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006).

[2] Shin, Y. J. et al. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep 5, 14269 (2015).

[3] Shen, H. et al. MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension. Circulation 142, 1190–1204 (2020).

[4] Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947–953 (2020).

Sequence and Features