UCSF

Synthetic peptide tags have been key to improving the ease with which scientists can interface with proteins in living systems. This PhD. work centers on demonstrating how peptide tags can be used to build modular platforms to accelerate live cell imaging, isolation of genetically engineered cells, cryo-EM, and applied DNA origami. First, we employed a set of three peptide/binder tagging systems, we developed tag-assisted splitHalo (TA-splitHalo), a versatile, multiplexable endogenous tagging platform. We applied TA-splitHalo sytems to live-cell imaging and complex knock-in sorting of lamin A/C knock-ins. Second, we utilized a peptide tag knock-in approach that combines split- fluorescent proteins and affinity purification to perform cryogenic electron microscopy (cryo-EM) on native structures derived from genetically edited suspension cell lines. This workflow was prototyped on native human proteasome complexes, demonstrating the pulldown of biologically relevant variants. Third and independently from the knock-in platforms, we engineered a SpyTag/SpyCatcher strategy in bacteria to create purification pipelines for proteins with two different and desired functional domains, enabling new ways to self-assemble biological nanoarchitectures. This was done by tethering cytokines to a transcription activator-like effector (TALE) protein to create immunomodulatory DNA binding proteins that can functionalize DNA origami structures. Our work demonstrates that the short length and minimally invasive nature of peptide tags enable plug-and-play platforms that can be applied at high-throughput for a wide range of applications.