Difference between revisions of "Part:BBa K4839023"

Revision as of 11:56, 12 October 2023

TRE3GS promoter- Anti- IRF4 BioPROTAC

This component consists of promoter PGK, downstream rtTA, and promoter TRE3GS, downstream Anti-IRF4 BioPROTAC. rtTA acts as a trans-activator in the Tet-inducible transcription system (Tet-ON system) and binds to the exogenous Dox to activate pTRE, initiating downstream transcription, and the downstream Anti-IRF4 BioPROTAC encoded product recognizes and binds IRF4 and induces its ubiquitinated degradation.

We have successfully constructed the IRF4 Binding protein (anti-IRF4 BioPROTAC) and assembled it into the pLVX-TREG3S-FLAG-TetOne-Puro vector regulated by doxycycline-controlled Tet-on system. We have also completed the construction of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and the sequencing results are shown in Figure 2.

Figure2. Sequencing results confirm the successful construction of IRF4 Binding protein (PU.1-SPOP).

The C-terminal E3 adaptor/E3 domain was selected based on the suggestion of Professor Ting Pan from Sun Yat-sen University School of Medicine. We chose SPOP, which has been widely used in previous studies, instead of using the search results from the database. We then designed the downstream experimental plan (Figure 1), aiming to induce the expression of anti-IRF4 BioPROTAC using doxycycline and achieve degradation of IRF4.

Figure1.A schematic proof of function for controllable degradation of IRF4 protein

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 2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.

Figure2. 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