Synthesis of proteins containing amino acids outside of the genetic code has recently resurfaced as a topic of much interest and presents a great challenge intellectually and technically. Reprogramming the genetic code requires two key events: (i) The creation of a new efficient 'orthogonal' aminoacyl tRNA synthetase:tRNA pair able to generate solely a non-canonical aminoacyl tRNA. (ii) The ability to specify the position in which this amino acid will be inserted in the protein by recoding a particular mRNA codon. Based on the natural existence of two archaeal aminoacyl-tRNA synthetase-like proteins (O-phosphoseryl tRNA synthetase with its cognate tRNACys and pyrrolysyl-tRNA synthetase with its cognate tRNAPyl) research will be initiated to develop an Escherichia coli system for the in vivo co translational insertion of phosphothreonine and phosphotyrosine at pre-determined positions in a polypeptide based on the nonsense codons UGA and UAG in a messenger RNA. Given the importance of phosphothreonine and phosphotyrosine in eukaryotic proteins essential for cell proliferation, cellular signaling, and cell death, a robust method of making proteins containing phospho-amino acids will have wide application and great significance. The broader impacts resulting from this project are: (i) The experimental tools to be developed will be widely employed in the future by other researchers in the life sciences. (ii) Development of a viable patent will contribute to commercial technology. (iii) This project will provide interdisciplinary training for undergraduate and postdoctoral students.
Proteins are key biomolecules in the cell mostly made up of twenty amino acid building blocks. Of special importance are the phosphoproteins that are responsible for most regulatory and signaling events in human cells. In these biomolecules certain amino acids carry a phosphate group which is added after the protein molecule is made. Currently there is no detailed understanding of the biosynthetic route to phosphoproteins and of the players involved in this process. Phosphoserine (Sep) is the most abundant phosphoamino acid. We developed an Escherichia coli based system for the co-translational insertion of phosphoserine at pre-determined positions in a protein based on the codon UAG in a messenger RNA (see Image). An engineered tRNA (tRNASep) that recognizes the codon UAG is attached to phosphoserine by the action of the enzyme SepRS. The resulting Sep-tRNASep is transported by an engineered elongation factor EF-Tu to the ribosome where Sep is inserted into the growing protein. Given the importance of phosphoserine in human proteins essential for cell proliferation, cellular signaling, and cell death, our robust method of making phosphoserine-containing proteins will have wide application and great significance. The broader impacts resulting from this work were four-fold: (i) the experimental tools developed in this study are being empoyed in over 100 laboratories by other researchers in the life sciences. (ii) A viable patent is contributing to commercial technology. (iii) This project furthered the training of a high school science teacher. (iv) Several minority undergraduate students participated in this research.
