Toolset for incorporation of unnatural amino acids into proteins expressed in mammalian cells

Proteins are biological nanomachines that perform almost all the biological processes. The survival of a living organism depends heavily on proper coordination and functioning of these biomolecules. Naturally, malfunctioning proteins may stunt the growth and proper development of an organism and might even lead to death. Because of their impact on human health, proteins are among the most studied biomolecules. For such studies, proteins are usually produced using protein expression systems (e.g., mammalian cells, insect cells, yeast cells or bacterial cells). For studying human proteins, mammalian systems or human cell- based systems are usually the best because they can most accurately mimic the production and maturation processes of the target-protein.
To understand the biophysical properties and working mechanisms of proteins, many biophysical methods are available. Such methods often rely on molecules known as probes, that can sense a change in their local environment and generate measurable signals corresponding to these changes. To study a protein molecule, these probes, must be covalently attached to the protein via one of its chemically reactive sites. Attachment of biophysical probe to a specific site on the protein requires a unique reactive site on the protein molecule. However, site-specific attachment of a biophysical probe is either not feasible or not practical when many reactive sites are present on the protein or if the reactive sites are functionally relevant for the protein. In such a scenario, presence of a bioorthogonal reactive handle on the protein of interest may allow the introduction of the biophysical probe without interfering with the reactive sites or functionally relevant amino acids of proteins. Such bioorthogonal reactive handles can be introduced in proteins via unnatural amino acids.
Using protein expression systems, unnatural amino acids can be genetically (or cotranslationally) incorporated into the proteins during the polypeptide biosynthesis. The most widely used strategy for doing this is genetic code expansion (G.C.E.) via stop codon suppression, where, by introducing an orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase pair in the protein expression system, one of the three stop-codons (Opal, Amber, or Ochre) is repurposed as a signal-codon to incorporate unnatural amino acid(s) in proteins. Several genetic code expansion systems have been developed to incorporate more than 100 different unnatural amino acids into the proteins expressed in mammalian systems. These unnatural amino acids provide a gamut of unique biophysical and biochemical characteristics for characterizing proteins and polypeptides. Among other applications, unnatural amino acids can introduce bioorthogonal reaction handles, cross-linking handles, post-translational modifications or fluorescent side chains into proteins.
This work is dedicated for site-specific incorporation of unnatural amino acids in proteins expressed in mammalian cells. For doing so, we have developed mammalian expression vectors and, in this thesis, have demonstrated their potential for assimilating the existing G.C.E. systems.
We have also developed a cell-based screening assay for quantification of the UAA incorporation efficiency of the different G.C.E. systems. We have demonstrated that this screening assay provides a holistic picture about UAA incorporation conditions. We have also demonstrated that this assay is compatible with high- throughput screening experiments.
Finally, we have demonstrated a systematic strategy for efficiently incorporating two UAAs in proteins expressed in mammalian cells. Using this strategy, we have incorporated a fluorescent amino acid as well as a bioorthogonal handle in our test protein eGFP. The introduced biorthogonal handle can be used for site-specific incorporation of a fluorophore to perform FRET based experiments.