Fig. 2: Optical activation of a Protease(Dap) substrate trap in human cells.
From: Mechanism-based traps enable protease and hydrolase substrate discovery
a, The mass of TEV(C151pc-Dap) before and after illumination of human cells expressing the protein. TEV containing pc-Dap was produced using the DapRS–tRNACUA pair and a TEV gene bearing the amber codon (TAG) at position 151 in the presence of pc-Dap. The grey trace shows proteins purified before illumination. Fully protected TEV(C151pc-Dap) bearing an acetyl group ([Ac-TEV(C151pc-Dap)]: expected 39,245.4 Da, observed 39,244.4 Da). The blue trace shows proteins purified immediately after illumination of cells. The fully deprotected product ([Ac-TEV(C151Dap)]: expected 38,948.2 Da, observed 38,945.2 Da); the deprotection intermediate ([Ac-TEV(C151Dapinter)]: expected 39,052.2 Da, observed 39,054.0 Da). The brown trace shows proteins purified 6 h after illumination of cells. b, TEV variants and GFP-s were co-expressed in HEK293T cells for 48 h. Total lysate was analysed by anti-Strep (for TEV) and anti-GFP antibodies. Wild-type TEV quantitatively cleaved GFP-s to GFP. β-Tubulin was used as a loading control. c, Detection of TEV(Dap)–GFP conjugate after Strep-tag enrichment. Samples, not illuminated (lane 4) or after UV illumination (lanes 1–3 and 5–10) were collected at indicated time points. Input, cell lysates before immunoprecipitation probed with anti-Strep and anti-GFP antibodies. β-Tubulin was used as a loading control. Experiments in a–c were performed in two biological replicates with similar results. For gel source data, see Supplementary Fig. 1.
