Figure 3.

MaPylRS supports in vivo synthesis of a protein containing a single β²-HA. (A) Workflow for protein expression in C321.ΔΑ.exp E. coli transformed with pMega-MaPylRS and pET22b-sfGFP-3TAG. (B) Plot of F₅₂₈/OD₆₀₀ values measured 24 h after induction with 1 mM IPTG as a function of substrate identity and concentration. n = 2 biological replicates, 3 technical replicates per biological replicate. (C) SDS-PAGE of sfGFP-3TAG expressed in the presence of 1–4. (D) Deconvoluted mass spectrum of sfGFP-3TAG expressed in the presence of (S)-β²-OH 3. (E) MS/MS profile of peptide fragment XSKGEE (where X is (S)-β²-OH 3) observed after GluC digestion of sfGFP-3TAG expressed in the presence of (S)-β²-OH 3. During collision dissociation, the Boc group of 3 is lost, and subsequent fragmentation generates the commonly observed b and y ions. We note that the analogous peptide fragment corresponding to incorporation of Gln at position 3 (AQSKGEE) is too hydrophilic to be retained on the column and cannot be quantified via LC-MS/MS. (F) Evaluation of the fidelity of incorporation of (S)-α-NH₂1, (S)-α-OH 2, or (S)-β²-OH 3 at position 3 of sfGFP-3TAG using a larger GluC digestion product that could be quantified. Here, X denotes position 3, and the preceding A is included when growths included (S)-α-NH₂1, Gln, Lys, or Tyr (as the products contain only amide bonds) and not when growths included (S)-α-OH 2 or (S)-β²-OH 3 (as the single ester bond undergoes hydrolysis). Analysis of this longer fragment reveals that more than 99% of the sfGFP-3TAG produced from growths supplemented with (S)-α-NH₂1 or (S)-α-OH 2 contains the requisite monomer at position 3. When the growths are supplemented with (S)-β²-OH 3, 63% of the sfGFP-TAG produced contains this monomer at position 3. The remaining material contains Gln (28.6%), Lys (4.4%), or Tyr (2.9%).