Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

Abstract

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Fig. 1.

Protocol for screening libraries of mutant aaRS. Cells transformed with an aaRS library are induced to express OmpC in medium supplemented with a noncanonical amino acid bearing a reactive side chain. Cells that successfully incorporate the noncanonical amino acid display the reactive side chain on their surfaces. Labeled cells are covalently tagged by a biotin probe and subsequently stained with fluorescent avidin. Fluorescence-activated cell sorting isolates the tagged cells from the remainder of the library. Sorted cells may be subjected to additional rounds of screening or analyzed immediately.

Fig. 2.

Noncanonical amino acids and tagging reagents used in this study. (A) 1, AHA; 2, ANL; 3, Tris(triazolyl)amine ligand for copper-catalyzed azide–alkyne ligation; 4, biotin–PEO–propargylamide; 5, biotin–PEO–cyclooctyne. (B) Scheme for biotin tagging of azide-functionalized E. coli cell surfaces. 1 or 2 is incorporated into OmpC to display the azide on the cell surface. Cell surface azides react either by Cu-catalyzed azide–alkyne ligation (top route) or by strain-promoted azide–alkyne ligation (bottom route).

Fig. 3.

Comparison of the extent of cell-surface labeling by Cu-catalyzed azide–alkyne ligation and strain-promoted azide–alkyne ligation. Cells displaying OmpC containing AHA were biotinylated either by means of Cu-catalyzed azide–alkyne ligation (50 μM 4, 200 μM 3, and 100 μM CuBr at 4°C) (Left) or by strain-promoted azide–alkyne ligation (100 μM 5 at 37°C) (Right). Data are plotted as forward scatter (FSC; x axis), which is an indication of cell size, vs. fluorescence (FL1; y axis). The median fluorescence of cells labeled by means of Cu-catalyzed azide–alkyne ligation is 3-fold larger than that of cells labeled by means of the strain-based reaction.

Fig. 4.

Residues in the Met-binding pocket of MetRS selected for saturation mutagenesis. Four residues (stick models) found within 4 Å of the sulfur atom or methyl group of Met (space-filling model) were mutated to all other possible amino acids. Figure was drawn from coordinates in ref. (Protein Data Bank ID code 1F4L).

Fig. 5.

Fluorescence histograms of cells induced to produce OmpC in medium supplemented with ANL. All cells were biotinylated by means of Cu-catalyzed azide–alkyne ligation before flow cytometry. (A) Cells harboring naïve MetRS library. (B) Cells enriched from the top 1% of cells in A. (C) Cells harboring clone 2.6.1 MetRS. (D) Cells harboring clone 2.6.2 MetRS.

Fig. 6.

SDS/PAGE analysis of whole-cell lysates of M15MA[pAJL-61] encoding the L13G MetRS mutant. (Lane 1) Molecular weight marker. Cells were grown to midlog phase in medium containing all 20 canonical amino acids and then shifted to 19-aa medium (no Met) supplemented with 40 mg/liter Met (lane 2), 172 mg/liter ANL (lane 3), or no amino acid (lane 4). Expression of DHFR is comparable in the cultures supplemented either with Met or ANL.