Figure 2.

Identifying fragmented MetRS variants that metabolically label proteins with Anl. (a) DNA that contains a terminator, promoter (P_T7), and ribosomal binding site was randomly inserted into the MetRS gene to create a library of vectors that express two-piece MetRS variants⁸. (b) Growth of E. coli CS50-DE3 cells in the absence of Met is compared for cells that express a functional MetRS truncation comprised of residues 1-548 and split MetRS variants. Ten-fold serial dilutions of cells were spotted on M9-agar plates and growth was imaged after 24 h. (c) Complementation strengths of fragmented MetRS variants, which were named based on the last residue encoded by their N-terminal fragments. (d) Color coding of the connective polypeptide [CP, blue], Rossmann fold [R, green], KMSKS [K, orange], and anticodon-binding [AB, purple] domains maps the locations of backbone fission (yellow spheres) in functional variants onto the MetRS structure¹¹. (e) Structure of Anl (1), which can be detected in proteins after reaction with alkyne-functionalized tetramethylrhodamine (TAMRA) dye (2). Copper-catalyzed azide-alkyne cycloaddition (3) results in a stable triazole linkage. (f) Cells expressing the split NLL-MetRS and PLL-MetRS from the vectors selected using bacterial complementation were grown in the presence of Anl, and cell lysates were treated with TAMRA-alkyne dye. In-gel fluorescence image shows TAMRA labeling, which indicates Anl incorporation into cellular proteins. Colloidal blue staining (bottom) was performed to compare protein loading across lanes.