Expanding the Genetic Code of Escherichia coli with Phosphoserine

Abstract

O -Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research.

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

“…It is not surprising; therefore, that dysfunctional signaling is thought to drive major human diseases including cancers and neurodegeneration [49,50]. The ability to synthesize sitespecifically phosphorylated proteins, such as phosphorylated Ub or recombinant active kinases [13], enables drug-screening efforts to explore new targets and to specifically target pathological phosphorylation [51]. We established an efficient route to pure recombinant phosphoprotein, and we have demonstrated that phosphoprotein purity is essential when enzyme activity depends critically on the site and occupancy of phosphorylation.…”

“…Previous reports have shown it is possible to produce designer phosphoprotein in E. coli [13,26], but the efficient production of pure phosphoprotein has remained challenging. We successfully produced several pUb variants in unambiguously pure form as evidenced by MS/MS and Phos-tag gels.…”

“…phosphorylated recombinant protein by reassigning the UAG codon to pSer [13]. The system requires a phosphoseryl-tRNA synthetase (SepRS), derived from an archaeal pathway for Cys-tRNA Cys synthesis, a mutant tRNA Sep [14], and a mutant elongation factor (EF-Sep) [13]. The pSer system is toxic to normal E. coli strains [15] and initial experiments showed phosphoprotein production was possible but inefficient (~1 lgÁL À1 culture) [15].…”

“…phosphorylated recombinant protein by reassigning the UAG codon to pSer [13]. The system requires a phosphoseryl-tRNA synthetase (SepRS), derived from an archaeal pathway for Cys-tRNA Cys synthesis, a mutant tRNA Sep [14], and a mutant elongation factor (EF-Sep) [13].…”

Rewiring translation – Genetic code expansion and its applications

“…Recently, Soll and colleagues succeeded in expanding the genetic code of E. coli with phosphoserine (11) [55]. They made use of the fact that a phosphoseryl-tRNA synthetase (SepRS) is involved in cysteine production in archeas.…”