Of GFP Y39TAG obtained by using AcRS in the SMER28 site absence of pAcPhe, no peptide signals corresponding to GFP sequence containing tyrosine or phenylalanine at position 39 were detected, indicating that similarly to the observed in vivo process [17,27] misacylation of suppressor tRNA by near-cognate endogenous amino acids was inhibited by addition of cognate UAA. Addition ofhigher concentrations of the UAA to the reaction, eliminate the competition fully, as no molecules with m/z equivalent to trypsin digested WT GFP were found for proteins obtained by addition of UAAs to the cell-free reaction. Moreover, no peptides containing canonical amino acids from near-cognate suppression of stop codon were detected, confirming good selectivity and fidelity of the analyzed M. 14636-12-5 jannaschii aaRSs. MS/MS analysis of GFP Y39TAG peptides (Fig. 4B, 5B and 6B) demonstrated a characteristic mass shift of 88.11, 109.9 and 26.04 Da relative to WT GFP, values that exactly match the differences between tyrosine and pBpa, pIPhe and pAcPhe, respectively. Mass shifts were also detected for peaks corresponding to “y” ions (from y3 to y14), as well as for b13 ion, indicating the site of UAAs incorporation to be position 39 of GFP. In general, we did not detect any differences in the GFP Y39TAG proteins obtained by utilization of either MjtRNACUA or tRNACUAOpt, apart from the different protein yields.DiscussionSince it has been shown that protein synthesis is a ribosomemediated process that does not require cell integrity, cell-free protein expression systems have been used for a variety of purposes, including site-specific UAA incorporation into recom-In-Vitro Translation with Unnatural Amino AcidsFigure 6. Site-specific pAcPhe incorporation into GFP in a cell-free expression system. (A) Western blot visualization of WT GFP and pAcPhe-incorporating GFP Y39TAG. The synthesis of GFP was performed using an in vitro translation kit. Co-translational incorporation of pAcPhe was achieved upon addition of the corresponding plasmid (500 mg/mL), M. jannaschii pAcPheRS (100 mg/mL), different types of suppressor tRNA (480 mg/mL), and pAcPhe (1 mM) to the reaction medium. tRNA denotes synthetic MjtRNACUA, Opt ?tRNACUAOpt. (B) Annotated average MS/MS spectrum of peptides with m/z 765.84 corresponding to pAcPhe-incorporated FSVSGEGEGDATY*GK peptide of GFP Y39TAG. The Y* ion corresponding to pAcPhe (calculated m/z, 393.24; observed m/z, 393.23) can be easily detected. The distinguishing mass shift of 26 Da can be observed for most abundant “y” ions. For clarity, only the most abundant “y” ions are assigned. doi:10.1371/journal.pone.0068363.gbinant proteins [21,26]. In our efforts to develop an E. coli-based cell-free protein expression system to produce high yields of UAA containing recombinant proteins, we employed the most established M. janaschii orthogonal synthetases and nonsense suppressor molecules. In contrast to previously described cell-free expression systems adapted for protein synthesis with encoded UAAs, we employed purified MjTyrRS and its derivatives along with suppressor tRNA as additional components of defined reaction mixture allowing for 23977191 control of the protein synthesis environment. In this manner, we were able to produce GFP Y39TAG with tyrosine with an absolute yield of 270 mg/mL and a suppression efficiency of 55 by adjusting MjTyrRS and MjtRNACUA concentrations. Although suppressor tRNA concentration is the major factor limiting production of proteins conta.Of GFP Y39TAG obtained by using AcRS in the absence of pAcPhe, no peptide signals corresponding to GFP sequence containing tyrosine or phenylalanine at position 39 were detected, indicating that similarly to the observed in vivo process [17,27] misacylation of suppressor tRNA by near-cognate endogenous amino acids was inhibited by addition of cognate UAA. Addition ofhigher concentrations of the UAA to the reaction, eliminate the competition fully, as no molecules with m/z equivalent to trypsin digested WT GFP were found for proteins obtained by addition of UAAs to the cell-free reaction. Moreover, no peptides containing canonical amino acids from near-cognate suppression of stop codon were detected, confirming good selectivity and fidelity of the analyzed M. jannaschii aaRSs. MS/MS analysis of GFP Y39TAG peptides (Fig. 4B, 5B and 6B) demonstrated a characteristic mass shift of 88.11, 109.9 and 26.04 Da relative to WT GFP, values that exactly match the differences between tyrosine and pBpa, pIPhe and pAcPhe, respectively. Mass shifts were also detected for peaks corresponding to “y” ions (from y3 to y14), as well as for b13 ion, indicating the site of UAAs incorporation to be position 39 of GFP. In general, we did not detect any differences in the GFP Y39TAG proteins obtained by utilization of either MjtRNACUA or tRNACUAOpt, apart from the different protein yields.DiscussionSince it has been shown that protein synthesis is a ribosomemediated process that does not require cell integrity, cell-free protein expression systems have been used for a variety of purposes, including site-specific UAA incorporation into recom-In-Vitro Translation with Unnatural Amino AcidsFigure 6. Site-specific pAcPhe incorporation into GFP in a cell-free expression system. (A) Western blot visualization of WT GFP and pAcPhe-incorporating GFP Y39TAG. The synthesis of GFP was performed using an in vitro translation kit. Co-translational incorporation of pAcPhe was achieved upon addition of the corresponding plasmid (500 mg/mL), M. jannaschii pAcPheRS (100 mg/mL), different types of suppressor tRNA (480 mg/mL), and pAcPhe (1 mM) to the reaction medium. tRNA denotes synthetic MjtRNACUA, Opt ?tRNACUAOpt. (B) Annotated average MS/MS spectrum of peptides with m/z 765.84 corresponding to pAcPhe-incorporated FSVSGEGEGDATY*GK peptide of GFP Y39TAG. The Y* ion corresponding to pAcPhe (calculated m/z, 393.24; observed m/z, 393.23) can be easily detected. The distinguishing mass shift of 26 Da can be observed for most abundant “y” ions. For clarity, only the most abundant “y” ions are assigned. doi:10.1371/journal.pone.0068363.gbinant proteins [21,26]. In our efforts to develop an E. coli-based cell-free protein expression system to produce high yields of UAA containing recombinant proteins, we employed the most established M. janaschii orthogonal synthetases and nonsense suppressor molecules. In contrast to previously described cell-free expression systems adapted for protein synthesis with encoded UAAs, we employed purified MjTyrRS and its derivatives along with suppressor tRNA as additional components of defined reaction mixture allowing for 23977191 control of the protein synthesis environment. In this manner, we were able to produce GFP Y39TAG with tyrosine with an absolute yield of 270 mg/mL and a suppression efficiency of 55 by adjusting MjTyrRS and MjtRNACUA concentrations. Although suppressor tRNA concentration is the major factor limiting production of proteins conta.