A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli

Abstract

To site-specifically incorporate an unnatural amino acid (UAA) into target proteins in Escherichia coli, we use a suppressor plasmid that provides an engineered suppressor tRNA and an aminoacyl-tRNA synthetase (aaRS) specific for the UAA of interest. The continuous drive to further improve UAA incorporation efficiency in E. coli has resulted in several generations of suppressor plasmids. Here we describe a new, highly efficient suppressor plasmid, pUltra, harboring a single copy each of the tRNA and aaRS expression cassettes that exhibits higher suppression activity than its predecessors. This system is able to efficiently incorporate up to three UAAs within the same protein at levels up to 30% of the level of wild-type protein expression. Its unique origin of replication (CloDF13) and antibiotic resistance marker (spectinomycin) allow pUltra to be used in conjunction with the previously reported pEVOL suppressor plasmid, each encoding a distinct tRNA/aaRS pair, to simultaneously insert two different UAAs into the same protein. We demonstrate the utility of this system by efficiently incorporating two bio-orthogonal UAAs containing keto and azido side chains into ketosteroid isomerase and subsequently derivatizing these amino acid residues with two distinct fluorophores, capable of Förster resonance energy transfer interaction. Finally, because of its minimal composition, two different tRNA/aaRS pairs were encoded in pUltra, allowing the generation of a single plasmid capable of dual suppression. The high suppression efficiency and the ability to harbor multiple tRNA/aaRS pairs make pUltra a useful system for conducting single- and multiple-UAA mutagenesis in E. coli.

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

“…The availability of such a polyspecific aaRS for use in mammalian cells would be attractive, allowing a single construct to be used for the incorporation of a number of distinct UAAs. The wild-type Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS) already exhibits significant polyspecificity, incorporating UAAs with bioorthogonal functional groups for conjugation reactions, photoaffinity probes, posttranslational modifications, and others (1,17). To identify additional polyspecific tRNA/aaRS pairs, we screened eight available E. coli TyrRS (EcTyrRS) variants, previously evolved in yeast for the incorporation of different UAAs (1).…”

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

“…For site-specific incorporation of Tby into proteins, we chose the p-cyanophenylalanyl-tRNA synthetase (pCNF-RS) evolved from the Methanocaldococcus jannaschii tyrosyl-RS encoded in the pUltra system together with the requisite tRNA CUA for recognition of TAG stop codons. 11 Bacillus stearothermophilus (Bst) DnaB was produced in fusion with the solubility enhancer GB1 by in vivo expression in Escherichia coli (Supporting Information Figures S1 and S2A), whereas Staphylococcus aureus sortase A (SrtA) and the E. coli aspartate/glutamate binding protein (DEBP) were produced from linear PCR amplified DNA in a cell-free system based on an E. coli cell extract, from which the release factor 1 had been selectively removed by tagging with chitin-binding domains and filtration over chitin ( Figure S2B). 12 The fusion with GB1 did not significantly change the 1D 1 H NMR spectrum of the Bst DnaB hexamer ( Figure S3A).…”

Recombinant thiopeptides containing noncanonical amino acids

“…(PylRS-OptBC) was used to enable efficient transcription in the low-GC-content Bacillus host (22). PylRS-OptBC was expressed under the transcriptional control of the sarA promoter [a global regulator of virulence in S. aureus (23)] or of the veg promoter (which is constitutively expressed during vegetative growth in B. subtilis).…”