Engineering Phosphoproteins in E. Coli

Protein kinases post-translationally phosphorylate specific residues as a means of regulating cell-signaling cascades. O-phosphoserine (Sep) being, by far, the most common phosphorylation modification. Phosphorylation events often require specific stimuli or conditions that are difficult to experimentally replicate, limiting investigations into the functional roles of these phosphoamino acids. Jesse Reinhart's group at Yale has devised a system to incorporate Sep directly into the genetic code of E. coli using a re-engineered tRNA (tRNAsep). This process requires not only the orthogonal tRNAsep:Sep-tRNA synthetase pair, but also a modified EF-Tu, which allows tRNAsep to be incorporated during protein synthesis. This study elegantly deduces the minimal requirements for genetic code expansion as well as provides a unique tool for protein engineering and research.  More information and the necessary plasmid reagents cna be found at Addgene, a non-profit plasmid repository.