Figure 4. Site-specific Bpa incorporation into GFP in a cell-free expression system.

(A) Western blot visualization of WT GFP and Bpa-incorporating GFP Y39TAG. The synthesis of GFP was performed using an in vitro translation kit. Co-translational incorporation of Bpa was achieved by addition of M. jannaschii BpaRS (100 µg/mL), different types of suppressor tRNA (480 µg/mL) and Bpa (1 mM) to the reaction medium. tRNA denotes synthetic MjtRNA_CUA, Opt – tRNA_CUA ^Opt. (B) Annotated MS/MS spectrum of the FSVSGEGEGDATY*GK peptide from GFP Y39Bpa. The mass shift of 88 Da between Bpa in GFP Y39Bpa and tyrosine in WT GFP is clearly observed. For clarity, only the most abundant “y” ions are assigned.