Novel tools for the study of protein-protein interactions in pluripotent cells

Abstract

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described.