Genetic Code Expansion in Mammalian Cells
Creators: name: Beranek, Vaclav
Publisher: name: Apollo - University of Cambridge Repository
Year: 2019
Subjects: Genetic code expansion
Phosphorylation
Mammalian Cells

description: Proteins in nature are synthesized from a conservative set of 20 canonical amino acids, limiting the chemical space of biological systems. Over the last few decades, scientists have developed methods to expand the genetic code of living organisms, introducing new, non-canonical amino acids with diverse chemistries into proteins. These methods rely on engineering of the translational machinery often importing an aminoacyl-tRNA synthetase (aaRS)/tRNA pair that does not cross-react with the endogenous aaRS/tRNA pairs. This approach has allowed for co-translational incorporation of a variety of non-canonical amino acids, including amino acids for photocrosslinking, biophysical probes, amino acids bearing post-translational modifications or bio-orthogonal chemical handles for protein labelling and imaging. These designer amino acids allow researchers to probe, image and control protein function in vivo with great precision and minimal perturbation. Cultured mammalian cells present an attractive model for studying human biology and disease. The first aaRS/tRNA pair used for genetic code expansion in mammalian cells was the Escherichia coli (Ec) tyrosyl-tRNA synthetase (TyrRS) and Bacillus stearothermophilus (Bs) tyrosyl-tRNA (tRNATyr), however, this pair was quickly supplanted by Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase/tRNA pairs (PylRS/tRNAPyl), which are easily engineered to incorporate a wide variety of useful non-canonical amino acids. The developments in the genetic code expansion in mammalian cells have allowed researchers to study role of post-translational modifications, dissect signalling pathways, label and identify cell proteomes and identify protein-protein interactions directly in vivo. This thesis presents several key advances in, and applications of, genetic code expansion in mammalian cells. Chapter 1 introduces the relevant aspects of protein translation and summarises the progress in the field of genetic code expansion to date. Chapter 2 describes genetic encoding of phosphoserine and its non-hydrolyzable analogue in mammalian cells. The engineered phosphoseryl-tRNA synthetase/tRNA pair (SepRSv1.0/tRNAv1.0CUA) derived from Methanococcus mariplaudis and Methanocaldococcus janaschii (Mj) is shown to be orthogonal in mammalian cells. Subsequently, phosphoserine incorporation into a reporter protein is optimised by engineered translation elongation factor 1 alpha, engineered eukaryotic release factor 1 and metabolic engineering of the mammalian cell line. Overall this approach achieves an order of magnitude improvement in protein yield over unoptimised system. Incorporation of non-hydrolysable phosphonate analogue of phosphoserine in an engineered cell line is subsequently demonstrated and used for synthetic activation of a protein kinase. Chapter 3 describes two methodological advances in the use of genetic code expansion for protein imaging. Firstly, we demonstrate the use of genetic code expansion for super-resolution microscopy. Secondly, we identify aberrantly extended endogenous proteins ending with the amber stop codon as a source of non-specific labelling. We then proceed to minimise this background by optimisation of the labelling protocol and use the resulting protocol for live-cell imaging of a recently discovered microprotein. Chapter 4 experimentally demonstrates the orthogonality of a recently discovered PylRS/tRNAPyl pair from Methanomethylophilus alvus (Ma) in mammalian cells. We further demonstrate that this pair is mutually orthogonal to the widely used MmPylRS/tRNAPyl pair, establishing a new, orthogonal pair for genetic code expansion in mammalian cells. The two pairs are used to site-specifically direct two distinct amino acids into a reporter protein.