CHEMBIOCHEM FULL PAPERS DOI: 10.1002/cbic.201402033

Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes Christoph Lammers, Liljan E. Hahn, and Heinz Neumann*[a] Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this

system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.

Introduction Apart from a few minor variations, the genetic code is universally conserved across all organisms. Efforts to synthetically expand the repertoire of genetically encoded amino acids have yielded a large toolset for protein engineering. This is achieved by the expression of an evolved orthogonal tRNA/ aminoacyl-tRNA synthetase (AARS) pair, of which the tRNA is modified to decode nonsense or frame-shift codons, and the amino acid specificity of the AARS is altered to recognize an unnatural amino acid (UAA). This approach has been employed to expand the genetic code of prokaryotes[1] and eukaryotes, in yeast,[2] mammalian cells,[3] and whole animals,[4] thus enabling the genetic installation of amino acids containing bioorthogonal chemical handles, photoreactive side chains, or post-translational modification.[5] The ability to equip proteins site-specifically with such functionalities has facilitated numerous elegant biological studies. For example, the pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/ PylT) pair from Methanosarcina barkeri has been adapted to encode N(e)-acetyl-l-lysine,[6] thus enabling the production of acetylated histones, which can be used to investigate the functional impact of this important post-translational modification on chromatin dynamics and gene expression.[7] UV-activatable crosslinker amino acids, such as p-benzoyl-l-phenylalanine, have been used extensively to study protein–protein interactions, and have uncovered the mechanistic principles underpinning important biological processes, such as the transport [a] C. Lammers, L. E. Hahn, Prof. Dr. H. Neumann Free Floater (Junior) Research Group “Applied Synthetic Biology” Institute for Microbiology and Genetics, Georg-August University Gçttingen Justus-von-Liebig Weg 11, 37077 Gçttingen (Germany) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402033.

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of lipopolysaccharides across the periplasm of Gram-negative bacteria[8] and the condensation of chromosomes during mitosis.[9] The installation of bio-orthogonal reactive groups has been used for labeling proteins with fluorescent dyes, thereby facilitating the study of protein folding[10] and conformational dynamics.[11] However, there are certain limitations. In bacteria, release factor 1 (RF1) competes with the suppressor tRNA for binding to amber codons in the A-site, thereby causing premature termination of translation. This is compounded when more than one UAA is incorporated into the same protein. Escherichia coli strains with reduced RF1 activity can be used to increase the efficiency of UAA incorporation.[12] Despite its importance in translation, RF1 was successfully deleted, either after replacing the amber stop codon in several essential genes with the ochre codon,[13] or in the background of an RF2 variant with enhanced UAA termination activity.[14] This permitted the incorporation of multiple identical UAAs into the same protein. Recently, multiplex automated genome engineering (MAGE)[16] was used to remove all amber codons from the E. coli genome; RF1 was deleted, and AARS/tRNACUA pairs were used to recode this codon.[15] These are significant achievements to facilitate the incorporation of multiple identical UAAs into a protein. The installation of multiple different UAAs into the same protein requires the combination of mutually orthogonal AARS/tRNA pairs directed to different codons. Suppression of two different stop codons (amber and ochre) allowed the incorporation of two distinct UAAs, by using Methanococcus jannaschii tyrosyl-tRNA synthetase (MjYRS) and Methanosarcina barkeri PylRS (MbPylRS), respectively.[17a] These tools facilitated the production of dual-labeled proteins for FRET studies.[17] The number of different stop codons available limits this approach ChemBioChem 0000, 00, 1 – 6

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CHEMBIOCHEM FULL PAPERS to two distinct UAAs. Further expansion of the genetic code therefore requires an alternative type of codon. Quadruplet codons might provide 256 (44) new blank codons, which could be used to encode entirely new types of polypeptides. However, quadruplet decoding by natural ribosomes is inherently very inefficient, thus limiting its use in standard genetic code expansion strategies.[18] “Orthogonal ribosomes” recognize an altered Shine–Dalgarno sequence, thus constituting a parallel and independent translational mechanism that is independent of the essential function of producing the cellular proteome.[19] This permitted their evolution towards acquiring altered decoding properties, which resulted in the creation of ribosome variants able to decode amber and quadruplet codons with significantly improved efficiency.[20] The original system developed by Chin and co-workers enabled the incorporation of azide and alkyne functional groups into genetically determined sites of the same protein.[20a] However, the presence of six exogenous components (two pairs of AARS/tRNA, an orthogonal RNA (omRNA) and an orthogonal ribosome (o-ribosome)) on four different plasmids severely slows growth of the host and limits potential applications. Carefully balanced expression of all components is desirable, as excessive production of a single component unnecessarily consumes biosynthetic energy and is likely to produce toxic side effects. Significant efforts have been made to optimize AARS and tRNA levels to maximize the yield of full-length protein.[17a, 21] However, no systematic analysis has been performed to correlate expression of AARSs and tRNAs with tRNA loading status (degree of acylation) and efficiency of incorporation. Here, we developed assays to measure the performance of the system, and we identified the MbPylRS level as a limiting factor for protein yield. By balancing the expression levels of individual components we were able to improve growth rates and protein yield five- to tenfold. These improvements are likely to increase the number of possible applications employing the incorporation of multiple different UAAs by orthogonal ribosomes.

Results and Discussion The original system used by Chin and co-workers to direct the incorporation of two distinct unnatural amino acids was designed with four different plasmids to encode two AARSs, two tRNAs, an orthogonal ribosome and a corresponding mRNA for the protein of interest.[20a] Unsurprisingly, cells harboring this number of plasmids (containing potentially toxic components) grow very slowly, thus limiting the yield and scope of this system. To identify the limiting components of the system we developed assays for the detection of aminoacyl-tRNA synthetases, tRNAs, and their loading status. We introduced His6 tags for detection, and potentially purification, of MbPylRS and M. jannaschii TyrRS* (MjYRS*, an engineered mutant that recognizes MjtRNAYUCCU).[20a] MjYRS* tolerated C-terminal tagging with His6 (no loss of activity, data not shown). However, addition of a His6 tag to either the N or C terminus of MbPylRS (on plasmid pBK-PylS) resulted in severe loss of activity, as determined by  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org the ability of the enzyme to suppress an amber codon in chloramphenicol acetyltransferase (CAT) in the presence of 1 mm N(e)-tert-butyl-oxycarbonyl-l-lysine (BocK), an efficient substrate analogue.[22] The weakly conserved linker region between the tRNA-binding domain at the N terminus and the Cterminal catalytic domain proved tolerant to the introduction of a His6 tag (Figures S1 and S2 in the Supporting Information). We therefore used this construct in further experiments. To detect the levels and loading status of the corresponding tRNAs, PylT and MjtRNAYUCCU, we designed digoxigenin-labeled hybridization probes for northern blot analysis (see the Supporting Information). Both tRNAs gave rise to a single band in the absence of the cognate AARS and substrate amino acid (Figure S3). Acylation of the tRNA resulted in a mobility shift in acid-urea gels (as described by Ambrogelly and co-workers),[23] thus facilitating the simultaneous quantification of the amount and loading status. Next, we quantified aminoacyl-tRNA synthetase levels at different stages of growth in rich medium. We observed a strong signal for MbPylRS during the early stages of exponential growth; this declined when the culture approached saturation (Figure S4 A). This indicates that the enzyme might become a limiting factor during prolonged induction of recombinant protein expression. By varying the expression level of MbPylRS (by using promoters of different strengths in pBK-PylS plasmids from a promoter library), we observed positive correlation between MbPylRS level, tRNA acylation, and suppression efficiency (Figure S4 B). Hence, MbPylRS level is the limiting factor in the performance of this system. When we combined pBK-PylS plasmids employing well-characterized promoters of different strength (Table S1) with pCDF-PylT-H3-R52TAG (encoding PylT and Histone H3 with an amber codon in place of Arg52), we found that a moderate increase in MbPylRS was beneficial, but that higher overexpression did not improve yields further (Figure S4 C). Stronger promoters might consume valuable biosynthetic resources, especially in cases when multiple components are introduced into a single cell. We therefore decided to use Pcon (a constitutive promoter) or Plac (IPTG inducible) to drive the expression of MbPylRS in further experiments. Similar experiments with MjYRS* levels did not show such a trend (Figure S5). To reduce the burden by plasmids and the antibiotics for their maintenance, we aimed to build a modular plasmid expressing both AARSs and tRNAs at well-controlled levels (Figure 1 A). In this plasmid each of the four parts is flanked by unique restriction sites, thus facilitating modular replacement of individual elements to create different combinations of AARS/tRNA variants. The first version of this plasmid was created with the (original) glnS promoter to drive the expression of both AARSs. We combined this plasmid with the evolved orthogonal ribosome Ribo-Q1, which has enhanced quadruplet- and amber-decoding properties,[20a] and an orthogonal mRNA encoding superfolded GFP (sfGFP) containing an amber codon (N150TAG) or a quadruplet codon and an amber codon (D134AGGA, N150TAG; Figure 1 B). Compared to the original system, which used four plasmids to encode all components, the new system ChemBioChem 0000, 00, 1 – 6

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Figure 2. Optimized system for the incorporation of two distinct UAAs by orthogonal translation. A) Growth and protein yield are improved by using the four-component plasmids. DH10B cells were transformed with Ribo-Q1, omRNA encoding sfGFP (AGGA/TAG), and different plasmids (encoding MbPylRS/PylT and MjYRS*/tRNAYUCCU) and grown in LB containing 1 mm BocK. Cell density (c) and in-cell sfGFP fluorescence (a) were monitored. B) Incorporation efficiency. DH10B cells harboring plasmids encoding o-GSTMBP (Y17AGGA, N234TAG), Ribo-Q1, and AARS/tRNA pairs as in Figure 1 A were grown in the presence of 1 mm BocK. Total protein was extracted; GST fusion proteins were batch purified by using GSH-beads. Ft, flow-through; P, pellet; E, eluate.

Figure 1. Improved plasmid system for the simultaneous suppression of quadruplet and amber codons by evolved orthogonal ribosomes. A) Modular four-component plasmid. B) sfGFP expression by orthogonal translation. E. coli DH10B cells were transformed with plasmids for Ribo-Q1, sfGFP omRNA (containing the indicated codons for UAA incorporation) and either two further plasmids (pCDF-PylS/T and pDULE-MjYRS*, “4 plasmids”) or one of three variants of the four-component plasmid to express two pairs of AARS/tRNA but differing in the promoter for the expression of MbPylRS. Cells were grown in LB medium supplemented with 1 mm BocK; in-cell sfGFP fluorescence was measured in a 96-well plate reader after normalization to OD600 = 0.5. A control (without sfGFP) was used to measure background fluorescence.

required only three. This resulted in a significantly improved growth rate and a slightly increased protein yield. The suppression efficiency of amber codons with the three-plasmid system containing the original glnS promoter was low. We therefore replaced the promoter with Pcon or Plac. This strongly improved amber suppression efficiency (five- to tenfold increase in protein yield compared to cells harboring four plasmids). Next, we tested the new plasmids for their impact on the growth of E. coli DH10B (Figure 2 A): cells transformed with four plasmids needed 24 h to achieve OD600 = 0.5 (no exponential growth phase), whereas cells containing the four-component plasmid displayed a doubling time of less than 2 h and  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

saturated at OD600 > 2. Simultaneously, we followed sfGFP expression photometrically. sfGFP fluorescence was barely above background in cells containing the four-plasmid system, but cells that contained the new plasmid (MbPylRS expression driven by Pcon or Plac) achieved much higher sfGFP expression. This was in contrast to the same plasmid with the glnS promoter (only an improvement in growth rate). Thus, by both optimizing MbPylRS expression and reducing the number of plasmids in the system we were able to improve recombinant protein yield five- to tenfold while simultaneously reducing the metabolic burden on the bacteria. To estimate the efficiency of suppression of quadruplet and amber codons we combined the different four-component plasmids with Ribo-Q1 and a plasmid encoding a fusion of glutathione-S-transferase (GST) and maltose binding protein (MBP) variant that contained a quadruplet (replacing Y17 of MBP) and an amber codon (replacing N234 of MBP) in E. coli DH10B (Figure 2 B). We were able to purify approximately 1 mg of full-length protein from 1 L of culture (AGGA translated as tyrosine, UAG translated as N(e)-tert-butyl-oxycarbonyl-llysine). The suppression efficiency of the quadruplet codon by the MjYRS*/tRNAYUCCU pair was determined as 20–30 %, by comparing the amount of the first truncation product (the result of a frame shift: AGGA decoded as arginine (AGG)) to the sum of the second truncation product and full-length protein. The amber codon was nearly quantitatively suppressed, by comparison of the amount of the second truncation product (the ChemBioChem 0000, 00, 1 – 6

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CHEMBIOCHEM FULL PAPERS result of termination by RF1 at the amber codon) to full-length protein. Next, we exchanged MjYRS* (in the four-component plasmid with Plac promoter driving MbPylRS expression) by a variant recognizing p-azido-l-phenylalanine (pAzF) and MjtRNAUCCU (see the Supporting Information). This allowed us to incorporate pAzF and N(e)-propargyl-l-lysine (PrK, another substrate of MbPylRS)[24] simultaneously into GST-MBP (Figure 3 A). This

Figure 3. Incorporation of pAzF and PrK into GST-MBP and labeling with fluorescent dyes. A) DH10B cells were transformed with plasmids encoding Ribo-Q1, o-GST-MBP (Y17AGGA, N234TAG), and one (Plac) or two plasmids encoding AARS/tRNA pairs to incorporate the indicated amino acids: labeled “3” or “4”, respectively. Cells were grown in media containing the corresponding UAAs. GST-MBP proteins were purified with GSH-beads and analyzed by SDS-PAGE. B) GST-MBP proteins were produced with the three-plasmid system, labeled with fluorescent dyes, and analyzed by SDS-PAGE and phosphoimaging.

resulted in significant increases in suppression efficiency and protein yield relative to the original four-plasmid system. The three-plasmid system (in the presence of 5 mm pAzF and 2 mm PrK) produced more protein from the same culture volume than the four plasmid system using tyrosine and 1 mm BocK. We estimated 100–200 mg of full-length protein from 1 L of culture, whereas no full-length protein was detectable with the four-plasmid system under the same conditions. The azide group of pAzF can be reacted with dibenzoyl-cyclooctyne-conjugated fluorophores in a strain promoted azide-alkyne coupling reaction; the alkyne group of PrK can be reacted with azide-conjugated fluorophores in a CuI-catalyzed [2+3] cycloaddition reaction.[25] Specific labeling of GST-MBP (pAzF/PrK) by these click reactions is shown in Figure 3 B.

Conclusions The improvements described here greatly increase the reliability and yield of protein produced by orthogonal ribosomes. We expect that future studies, for example on optimal expression of orthogonal ribosomes, will further enhance system performance. Combined with the identification of additional mutually orthogonal AARS/tRNA pairs[18a, 26] this should allow the exploration of additional blank codons for UAA incorporation and eventually the production of proteins with more than two distinct UAAs or entirely unnatural polymers. The analysis of  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org the limiting factors in this work improves our understanding of the requirements and might help such future developments.

Experimental Section Chemicals, plasmids, strains, and medium: Antibiotics used in this study: ampicillin (100 mg mL 1; AppliChem, Darmstadt, Germany), chloramphenicol (50 mg mL 1 unless stated otherwise; AppliChem), kanamycin (50 mg mL 1; AppliChem; 25 mg mL 1 for pSC101*-Ribo-Q1), spectinomycin (75 mg mL 1; Sigma–Aldrich), and tetracycline (25 mg mL 1; AppliChem). The protease inhibitor cocktail comprised pefabloc SC (75 mm), leupeptin (0.15 mm), o-phenanthroline (37.5 mm), and pepstatin A (0.5 mm; all purchased from Sigma–Aldrich). BocK was purchased from Bachem (Bubendorf, Switzerland), pAzF was from ChemImpex (Wood Dale, IL). PrK was synthesized as previously described.[24] Protein molecular weights were assessed with a PageRuler Prestained Protein Ladder (Thermo Scientific). Antibodies used in western and northern blots were mouse anti-His (final 10 mg mL 1; GE Healthcare), sheep anti-DIG-AP (75 mU mL 1 Roche), goat anti-mouse HRP (0.1 mg mL 1 Abcam). E. coli strains DH10B (Life Technologies) and BL21(DE3) (Merck Millipore) were grown in lysogeny broth (LB) supplemented with antibiotics and UAAs as indicated. Plasmid sequences can be found in the Supporting Information. Western blots: Proteins were separated by SDS-PAGE and transferred to PVDF membranes by semidry or wet blot. Membranes were stained with Ponceau S and blocked for 10 min with BSA (3 % in PBS). Primary antibodies were applied for 2 h with shaking at room temperature or overnight at 4 8C. The membranes were then washed (3  10 min) with PBS containing Tween-20 (0.1 % v/v). Secondary antibodies were incubated for 1 h at RT, and membranes were washed again with PBS ( 3). HRP signals were detected by chemiluminescence by using ECL substrates and Hyperfilm (GE Healthcare). Protein purification and labeling: For GST-MBP purification, overnight cultures of E. coli DH10B transformed with the appropriate plasmid combinations (pSC101*-Ribo-Q1, a plasmid for o-GST-MBP containing the indicated mutations for incorporation of UAAs and plasmids for the expression of AARS/tRNA pairs) were used to inoculate LB (50 mL) to OD600 = 0.1. Full-length protein expression was started after 3 h by the addition of UAAs (BocK (1 mm), or pAzF (5 mm) and PrK (2 mm)) and IPTG (1 mm, for Plac constructs). After 4 h, cells were pelleted, and batch purification was carried out with GSH beads (GE Healthcare) by published procedures.[20b] Proteins for SDS-PAGE analysis were eluted from beads by heating (80 8C, 10 min) in sample buffer; for labeling experiments proteins were eluted in PBS containing glutathione (30 mm; AppliChem). The alkyne moiety of PrK was modified in a CuI-catalyzed click reaction according to a previous method.[20a] The protein was dissolved in sodium phosphate buffer (50 mm, pH 8.3) and mixed with Alexa Fluor 647 azide (25 mol equiv; Invitrogen). The reaction was performed at RT for 15 min in the presence of ascorbic acid (1 mm), CuSO4 (1 mm), and o-phenantroline (2 mm), and then incubated for a further 1 h at 4 8C. The azido functionality of pAzF was modified in a copper-free click reaction with DBCO488 (50 mol equiv; Jena Bioscience, Jena, Germany) in sodium phosphate (50 mm, pH 8.3) for 1 h at RT. Samples were mixed with glycerol (10 %) and analyzed by 15 % SDS-PAGE; fluorescence bands were detected by a Typhoon 9400 Variable Mode Imager (Amersham/GE). In-cell GFP fluorescence measurements: Overnight cultures of E. coli DH10B (transformed with the appropriate plasmid combinaChemBioChem 0000, 00, 1 – 6

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CHEMBIOCHEM FULL PAPERS tions) were used to inoculate LB (20 mL) to OD600 = 0.1. Cells were grown at 37 8C; full-length protein expression was started after 3 h by addition of BocK (1 mm) and IPTG (1 mm, for Plac constructs). Cells (1 mL, normalized to OD600 = 0.5) were pelleted and resuspended in PBS (1 mL), and sfGFP fluorescence in live cells was quantified by using a Fluostar Omega 96-well fluorescence plate reader (BMG Labtech, Offenburg, Germany).

Acknowledgements We thank Jason W. Chin for materials and Miguel Loera Sanchez for help during initial stages of the project. Research in the laboratory of H.N. is funded by the German Research Foundation (Emmy-Noether Programme), the Freefloater-Programme of the University of Gçttingen, the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain and the Fonds der Chemischen Industrie. Keywords: amino acids · click chemistry · genetic code expansion · optimization · orthogonal ribosomes · unnatural amino acids [1] L. Wang, A. Brock, B. Herberich, P. G. Schultz, Science 2001, 292, 498. [2] a) J. W. Chin, T. A. Cropp, J. C. Anderson, M. Mukherji, Z. Zhang, P. G. Schultz, Science 2003, 301, 964; b) S. M. Hancock, R. Uprety, A. Deiters, J. W. Chin, J. Am. Chem. Soc. 2010, 132, 14819. [3] a) P. R. Chen, D. Groff, J. Guo, W. Ou, S. Cellitti, B. H. Geierstanger, P. G. Schultz, Angew. Chem. Int. Ed. 2009, 48, 4052; Angew. Chem. 2009, 121, 4112; b) A. Gautier, D. P. Nguyen, H. Lusic, W. An, A. Deiters, J. W. Chin, J. Am. Chem. Soc. 2010, 132, 4086; c) N. Hino, Y. Okazaki, T. Kobayashi, A. Hayashi, K. Sakamoto, S. Yokoyama, Nat. Methods 2005, 2, 201; d) W. S. Liu, A. Brock, S. Chen, S. B. Chen, P. G. Schultz, Nat. Methods 2007, 4, 239. [4] a) A. Bianco, F. M. Townsley, S. Greiss, K. Lang, J. W. Chin, Nat. Chem. Biol. 2012, 8, 748; b) S. Greiss, J. W. Chin, J. Am. Chem. Soc. 2011, 133, 14196; c) A. R. Parrish, X. Y. She, Z. Xiang, I. Coin, Z. X. Shen, S. P. Briggs, A. Dillin, L. Wang, ACS Chem. Biol. 2012, 7, 1292. [5] H. Neumann, FEBS Lett. 2012, 586, 2057. [6] H. Neumann, S. Y. Peak-Chew, J. W. Chin, Nat. Chem. Biol. 2008, 4, 232. [7] a) H. Neumann, S. M. Hancock, R. Buning, A. Routh, L. Chapman, J. Somers, T. Owen-Hughes, J. van Noort, D. Rhodes, J. W. Chin, Mol. Cell 2009, 36, 153; b) P. Tropberger, S. Pott, C. Keller, K. Kamieniarz-Gdula, M. Caron, F. Richter, G. Li, G. Mittler, E. T. Liu, M. Bhler, R. Margueron, R. Schneider, Cell 2013, 152, 859. [8] S. Okuda, E. Freinkman, D. Kahne, Science 2012, 338, 1214.

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FULL PAPERS C. Lammers, L. E. Hahn, H. Neumann* && – && Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes

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Engineered orthogonal translation systems: Production of proteins with multiple distinct unnatural amino acids by evolved orthogonal ribosomes requires the simultaneous expression of six components. Reduction of plasmid number and optimization of expression levels significantly improves growth rate and protein yield.

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Optimized plasmid systems for the incorporation of multiple different unnatural amino acids by evolved orthogonal ribosomes.

Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are e...
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