Fig. 3. Development of an efficient and selective MbPylRS/tRNA pair for site-specific incorporation of Bpa.

a, Crystal structure of PylRS (gray) with adenylated pyrrolysine (blue; Protein Data Bank ID 2Q7H). Residues N311, C313, W382 and W386 (orange) in MbPylRS were randomized to all combinations of amino acids for directed evolution experiments. b, To isolate Bpa-specific mutants of MbPylRS, three rounds of directed evolution were performed, and subsequently, 192 clones were screened (Supplementary Fig. 7). Two clones, A2 and E10, survived on chloramphenicol concentration up to 300 μg ml⁻¹ in the presence of 1 mM Bpa (A2 +Bpa and E10 +Bpa) but did not survive in the absence of Bpa on chloramphenicol concentration at and above 100 μg ml⁻¹ (A2 −Bpa and E10 −Bpa). Both clones were the same and had the following mutations compared with the wild-type MbPylRS: N311Q, C313T and W382A, in addition to the Y349F preprogrammed mutation. c, Expression of gst-1TAG-cam using MbPyl(Bpa)RS without ncAA, with 1 mM Bpa, 1 mM pcY, 1 mM AzF or 1 mM BocK. Control expression using wt-MbPylRS that is known to efficiently incorporate BocK was also performed. Comparison of band intensities of full-length Gst-CaM demonstrates that newly evolved MbPyl(Bpa)RS is highly efficient and specific at incorporating Bpa (Supplementary Fig. 8). These experiments were repeated twice with similar results. d, Selective incorporation of Bpa at positions 32 and 109 in 7D12 using MbPyl(Bpa)RS. Comparison of band intensities for expression with Bpa (1 mM), pcY (1 mM), and both Bpa (1 mM) and pcY (1 mM) indicates selective incorporation of Bpa in the presence of pcY. These experiments were repeated twice with similar results. e, ESI-MS of amber mutants of 7D12 expressed using MbPyl(Bpa)RS in the presence of both Bpa (1 mM) and pcY (1 mM) demonstrates that Bpa is selectively incorporated. The dotted lines with arrows indicate expected molecular masses if pcY was incorporated (Supplementary Fig. 9). For gel images, lanes marked L are the Invitrogen SeeBlue Plus2 Pre-stained Protein Standard (catalog no. LC5925), and lanes marked Lʹ are the Thermo Scientific PageRuler Unstained Low Range Protein Ladder (catalog no. 26632).

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