Part:BBa_K4839024
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
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 257
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2527
Illegal SpeI site found at 257 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2627
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 257
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 257
Illegal NgoMIV site found at 2052 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 99
Illegal BsaI.rc site found at 2358
Illegal SapI site found at 1510
Illegal SapI.rc site found at 1441
| None |