Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Abstract

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.click chemistry ͉ high-throughput screening ͉ protein engineering

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…75 Recently, additional MetRS mutant screening strategies have been employed to further improve efficiency and selectivity for NLL. 76 Overall, the simplicity of the expression protocols for incorporating these analogs, as well as practical modified protein yields, make this a 16 suitable method for fluorescent protein labeling either in vitro or in vivo.…”

Cell-specific proteomic analysis in Caenorhabditis elegans

“…Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1,2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing L-methionine (Met) analog L-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3,4). Because Anl is a poor substrate for any of the natural aminoacyl-tRNA synthetases, it is excluded from proteins made in wild-type cells but is incorporated readily into proteins made in cells that express an appropriately engineered MetRS.…”

Protein Engineering with Non-Natural Amino Acids

“…The mutant MetRS activated azidonorleucine two-fold more efficiently than Met. As a result, the target protein was expressed with azidonorleucine at higher levels than with Met (Tanrikulu et al, 2009). By utilizing auxotrophic host cells for two or more canonical amino acids, it may be possible to produce recombinant proteins with more than one type of NNAA by this strategy.…”

“…Alternatively, a monoclonal antibody that binds specifically to a NNAA-containing peptide may be used for selection (Pastrnak & Schultz, 2001); conjugation of a florescent tag to the NNAA (containing a special chemical reactive group) displayed on the cell surface, followed by cell sorting via flow cytometry, may also lead to selection of the aaRS or tRNA. In fact, MetRS mutants with improved activities toward azidonorleucine and better discrimination against Met were generated via surface labelling and cell sorting (Tanrikulu et al, 2009). The surface-display based selection can be applied to evolve aaRS/tRNA for either a sense codon or a stop codon.…”