Interfering with protein expression is a powerful strategy to investigate the function of a protein. TRIM-AWAY, a novel strategy through microinjecting antibody and introducing of Trim21 protein into cells, harnesses the cellular protein degradation machinery to remove unmodified native proteins and allows the study of protein function in diverse cell types. But its poor cell viability after microinjection or electroporation, the high cost of antibody production, the short lifespan of injected-antibody inside the cell and the technique complexity limit its wide application.
This year through combining TRIM-AWAY strategy and ectopic expression of nanobody, we developed a new method for degrading endogenous proteins in mammalian cells. Basically, parts for expression Trim21 and nanobody-IgG Fc were constructed, and then were inserted in a single vector to realize P2A mediated bicistronic expression. To prove the concept, sequences for GFP nanobody and human IgG Fc fragment expression were ligated for fusion expression. To demonstrate our design, the receptor tyrosine-protein kinase erbB-3 is selected. This protein is able to bind and form the heterodimerization with proteins from the erbB family, which is highly involved in proliferation and metastasis of cancer cells, also implicated in chemotherapeutic resistance through multiple signaling pathways. It has been proved that the downregulation of erbB-3 slows down the process several cancers. In our demonstration, the erbB-3 inhibitory antibody would be expressed along with trim21 in cells and directly functions on erbB-3 to bring down its containment.