PR PREDATOR-An improved protein degradation method based on ectopic expression of TRIM21 and recombinant antibody

Design:

By combining TRIM-AWAY strategy and ectopic expression of nanobody, we developed an improved method for degrading endogenous proteins in mammalian cells, named as PR PREDATOR.

Two major barriers occur when attempting to selectively degrade endogenous proteins, one of which is the specificity, and the second is a powerful degradation mechanism to rely on. To address the specificity concern, we managed to use existing nanobody technique to achieve highly specific target recognition. By scanning existing patent files and literature reports, we built a data sheet with all available nanobodies and their target proteins enclosed. This data sheet can provide a sound basis for standardization of these nanobodies for further usage. The nanobodies are then fused with human IgG-Fc domain to bind Trim21, a powerful E3 ubiquitin-protein ligase we chose to mediate the degradation of target protein through proteasome. The plasmid expressing the PR PREDATOR system was constructed by putting the coding regions of HA-Trim21 and nanobody-IgG Fc under CMV promoter. The HA-Trim21 and nanobody-IgG Fc regions were separated by P2A sequence to achieve bicistronic expression (Figure 1a).

We introduced the Recombinant plasmids into cells, nanobody specific binds to proteins with high affinity, forming tight complexes. Trim21 binds with high affinity to the Fc domain of antibodies and TRIM21 recruits the ubiquitin-proteasome system to antibody-bound complex, leading to their destruction.