Intracellular delivery of proteins for live cell imaging

The majority of cellular functions are regulated by intracellular proteins, and regulating their interactions can unlock fundamental insights in biology and open new avenues for drug discovery. Because the vast majority of intracellular targets remain undruggable, there is significant current interest in developing protein-based agents especially monoclonal antibodies due to their specificity, availability, and established screening/engineering methods. However, efficient delivery of proteins into the cytoplasm has been a major challenge in biological engineering and drug discovery. We previously reported a platform technology based on a Coomassie blue-cholesterol conjugate (CB-tag) capable of delivering small proteins directly into the cytoplasm. Here, we report a new generation of CB-tag that can bring proteins with a wide size range into the cytoplasm, bypassing endosomal sequestration. Remarkably, intracellular targets with distinct structures were visualized. Overall, the new CB-tag demonstrated a robust ability in protein delivery with broad applications ranging from live-cell immunofluorescence to protein-based therapeutic development.