Efforts in my laboratory continue to advance CD-tagging, a method for genomic and proteomic discovery that generates cell lines that express proteins tagged with GFP or other reporters. Unlike other approaches for generating reporter-tagged cell lines, CD-tagging generates cells in which native transcriptional regulation and alternative splicing of the tagged genes are preserved.
The CD-tagging approach enables:
1. Discovery of previously unknown or poorly characterized proteins,
2. Observation and quantitation of the location and abundance of individual protein species in living cells and tissues,
3. Purification of tagged transcripts and proteins for biochemical and/or functional analysis,
4. Discovery and analysis of posttranslational modifications and protein-protein interactions, and
5. Development of new cell based assays.
6. Current work in the laboratory is focused on production and analysis of CD-tagged clones in mouse and human cultured cell lines and in mouse embryonic stem cells (mES cells).
FAP-based Biosensor Development
We have had considerable success in developing new live-cell assays for membrane protein translocation that employ FAP-based biosensors. FAPs are a new class of genetically encoded fluorescent tag developed in the Molecular Biosensor and Imaging Center (MBIC) here at CMU. FAP tags exhibit fluorescence only when bound to certain soluble fluorogens such as thiazole orange or malachite green.
Current work is focused on tracking protein translocation to or from the plasma membrane for a number of G-protein coupled receptors (GPCRs), and for the cystic fibrosis transmembrane conductance regulator (CFTR) and associated proteins. Additional efforts are aimed at developing new biosensors of protein proximity in living cells.
On the left, a group of FAP-labeled cells glow orange in the presence of a fluorogen dye. After the cells are treated with an agonist that activates the G protein-coupled receptors, the receptors move to the inside the cell, leaving many fewer on the surface, as shown in the image on the right.