Synthetic Amino Acids Research Articles

Pointing in the Right Direction: Controlling the Orientation of Proteins on Nanoparticles Improves Targeting Efficiency.

Protein-conjugated nanoparticles have the potential to precisely deliver therapeutics to target sites in the body by specifically binding to cell surface receptors. To maximize targeting efficiency, the three-dimensional presentation of ligands toward these receptors is crucial. Herein, we demonstrate significantly enhanced targeting of nanoparticles to cancer cells by controlling the protein orientation on the nanoparticle surface. To engineer the point of attachment, we used amber codon reassignment to incorporate a synthetic amino acid, p-azidophenylalanine (azPhe), at specific locations within a single domain antibody (sdAb or nanobody) that recognizes the human epidermal growth factor receptor (EGFR). The azPhe modified sdAb can be tethered to the nanoparticle in a specific orientation using a bioorthogonal click reaction with a strained cyclooctyne. The crystal structure of the sdAb bound to EGFR was used to rationally select sites likely to optimally display the sdAb upon conjugation to a fluorescent nanocrystal (Qdot). Qdots with sdAb attached at the azPhe13 position showed 6 times greater binding affinity to EGFR expressing A549 cells, compared to Qdots with conventionally (succinimidyl ester) conjugated sdAb. As ligand-targeted delivery systems move toward clinical application, this work shows that nanoparticle targeting can be optimized by engineering the site of protein conjugation.

Characterization and Scaled-Up Production of Azido-Functionalized Silk Fiber Produced by Transgenic Silkworms with an Expanded Genetic Code.

The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F1 hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions.

Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing.

The CRISPR-Cas9 nuclease has been repurposed as a tool for gene repression (CRISPRi). This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-mediated repression has been achieved with inducible promoters that regulate the expression of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly leaky dcas9 expression leads to significant background levels of repression of the target gene. In this study, we report on the development of optimized control of dCas9-mediated silencing through additional regulation at the translation level. We have introduced the TAG stop codon in the dcas9 gene in order to insert a synthetic amino acid, l-biphenylalanine (BipA), at a permissive site in the dCas9 protein. In the absence of BipA, a nonfunctional, truncated dCas9 is produced, but when BipA is present, the TAG codon is translated resulting in a functional, full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-BipA revealed a 14-fold reduction in background repression by the latter system. The here developed proof-of-principle system thus reduces unwanted background levels of gene silencing, allowing for tight and timed control of target gene expression.

Gut microflora and intestinal morphology changes of broiler chickens fed reducing dietary protein supplemented with lysine, methionine, and threonine in tropical environment

ABSTRACT This experiment aimed to discover the effect of reducing dietary protein supplemented with lysine, methionine, and threonine on growth performance, volatile fatty acid profile, and intestinal villus height and crypt depth of broilers, as well as the microflora counts isolated from broiler chicken faeces. A total of 288-day-old broilers were allocated to eight treatments with six replicates consisting of 36 birds per treatment. The diets contained dietary protein from 21 to 18% in starter diet and 18 to 16% in finisher diet supplemented with L-lysine, DL-methionine, and L-threonine at the same ratio for all dietary treatments. Body weight and feed intake were determined, and feed conversion ratio was calculated. Blood, intestine, and digesta samples were collected at 21 and 42 days for further analysis. Dietary protein supplemented with amino acids improved growth performance, reduced pathogenic bacteria, and increased beneficial bacteria counts, small intestine villi height and crypt depth, and ileal-digesta volatile fatty acid concentrations of broiler chickens. However, reducing 2% of dietary protein supplemented with lysine, methionine, and threonine showed the best results, especially in growth performance, feed conversion ratio, microflora count, duodenal and jejunal villi height, and ileal-digesta volatile fatty acid concentrations, such as butyric and valeric acids. It is believed that by reducing the level of dietary protein in broiler diet while supplementing with synthetic amino acid may enhance the intestinal morphology, nutrient digestibility, and absorption in broiler chickens and will simultaneously result in better performance.

Effect of Supplementation of Synthetic Lysine and Methionine on Serum Biochemical Profile, Carcass Characteristics and Meat Composition in Broiler Chicken

A total of 144 day old broiler chicks of Vencobb strain 400 randomly distributed into four groups (3 replicates of 12 chicks) using randomized block design viz. T0-basal diet (control), T1, T2 and T3 were offered the basal diet supplemented with synthetic L-lysine at 20, 30 and 40% and DL-methionine at 30, 40 and 50% in excess of the total BIS (2007) recommendations upto 42 days of age. Non-significant effect of supplementation of synthetic lysine and methionine were observed on feed intake. However, total gain in body weight and FCR were significantly (P<0.01) influenced by supplementation. Significant increase (P<0.001) in serum total protein was observed in the birds of T2 group whereas serum cholesterol and triglyceride level decreased (P<0.01) with increased level of synthetic amino acids. Statistically non-significant effect of supplementation was observed on dressing percentage, yield of prime cuts, percent weight of visceral organs and chemical composition of meat except that the abdominal fat pad was lower in groups T2 and T3. The study revealed that supplementation of 30% synthetic L-lysine and 40% DL-methionine higher than BIS (2007) recommendation in broiler rations reduced serum cholesterol and triglyceride levels.

EFFECTS OF METABOLIZABLE ENERGY AND LYSINE ON GROWTH AND FEED CONVERSION RATIO OF H’MONG BROILERS AT 0 TO 4 WEEK - AGE

An experiment was conducted to evaluate the interactive influences of dietary ME and lysine on growth of H’mong broilers from 0 to 28 days of age. The study was in a 3x3 two-factorial design, with the first factor being 3 levels of ME (3,000; 3,100 and 3,200 kcal/kg of feed) and the second one being 3 levels of lysine (1.1%; 1.0% and 0.9%), and 4 replicates with 6 broilers for each. Two hundreds and sixteen broilers were placed into 36 bamboo floor pens (6 birds/pen). The diets were formatted from corn yellow, soybean meal, soybean oil, DCP, shell, mineral and vitamin premixes, salt, and some of synthetic essential amino acids. Chicks were fed ad libitum and provided water all of time. The feeding trial was lasted for 4 weeks. The result indicated that the ME 3,000 kcal/kg of feed and lysine 1.1% diet resulted in the most efficient feed conversion ratio.

Welfare Quality® assessment of a fast-growing and a slower-growing broiler hybrid, reared until 10 weeks and fed a low-protein, high-protein or mussel-meal diet

Animal welfare is an important aspect of organic broiler (OB) production which involves e.g. up to twice the rearing period compared with conventional production and feedstuffs without prophylactic additives or synthetic amino acids. As market demand for organic broiler meat increases, the formerly modest OB production in Sweden is now rapidly increasing and research is needed to support this development. It is essential to use genotypes that adapt well to the rearing environment, in order to achieve satisfactory animal welfare. Health problems related to fast growth rate are a common animal welfare issue in modern broilers and this applies to systems with long rearing periods. In this study, the Welfare Quality® assessment protocol for poultry was used for assessment of lameness, contact dermatitis, cleanliness, thermal comfort, litter quality and the human-animal relationship in two modern broiler genotypes; the fast-growing Ross 308 (R) and the slower-growing Rowan Ranger (RR). In total, 645 day-old chicks (328 R and 317 RR) were reared until 10 weeks of age, which is the rearing period formerly used in organic production in Sweden, and fed a low-protein (L), high-protein (H) or mussel-meal (M) diet containing 14.5%, 17.0% or 15.6% crude protein, respectively. Broiler welfare was assessed on three occasions (at weeks 2, 6 and 9). The results showed rapid deterioration in welfare for fast-growing broilers when kept beyond 6 weeks. Mortality rate and incidence of lameness and contact dermatitis increased and litter quality, thermal comfort and plumage cleanliness decreased. Indications of poor welfare were also observed in the slower-growing hybrid, but to a lesser extent and later during rearing. Diet type only had minor effects on bird welfare, although R birds grew faster on the M diet. Thus the slower-growing RR hybrid is preferable to the fast-growing broiler type in production systems with a long rearing period. However, the RR growth rate can be regarded as moderate and, to avoid health problems related to fast growth rate, hybrids that grow even more slowly should be considered for OB production systems with a long rearing period.

Gabapentin Can Suppress Cell Proliferation Independent of the Cytosolic Branched-Chain Amino Acid Transferase 1 (BCAT1).

The metabolism of branched-chain amino acids (BCAA) has recently been implicated in the growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces the growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branched-chain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branched-chain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to the lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1 and may affect mitochondrial BCKA catabolism.

Performance and economic benefits of meat-type chicken fed diets containing white and yellow cassava supplemented with different additives

Two hundred and forty unsexed day-old broiler chickens were allotted to eight dietary treatments arranged in a 4 × 2 factorial arrangement of two cassava varieties (white and yellow cassava) supplemented with no additive (control), synthetic amino acid, cellulase enzyme and a combination of amino acid and cellulase enzyme for the starter (0–4 weeks) and finisher (4–8 weeks) phases. Data was collected on growth performance, feed conversion ratio (FCR), cost of feed consumed per kilogram and analysed using ANOVA. Starting broilers fed a diet containing yellow cassava supplemented with amino acids had the most superior (p < 0.05) final weight (709.09 g/bird), weight gain (657.27 g/bird), best FCR (1.93), and the cheapest (p < 0.05) cost per kilogram weight gain (₦200.15/kg [US$0.56]). Finishing broilers fed a diet containing white cassava supplemented with amino acid also recorded the best (p < 0.05) FCR (2.43) and cheapest cost of feed consumed per kilogram weight gain (₦226.71/kg [$0.63]). The study concluded that dietary supplementation with amino acid when white or yellow cassava root is to be used in the nutrition of broilers is essential for improved growth performance and economic benefits to broiler farmers.

Properties, biosynthesis, and catalytic mechanisms of hydroxy-amino-acids

The hydroxy amino acids have its unique effects in the biotechnology and molecular biology, and all sorts of synthetic hydroxy amino acids have also been developed. The hydroxy amino acids have been proved to be valuable for antifungal, antibacterial, antiviral and anticancer properties and known as a constituent of pharmaceutical intermediates. In addition to these fundamental researches, the hydroxy amino acids are applied widely to the synthesis of chiral drugs, such as (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL) is proved to be worthy in medical treatment, cis-4-hydroxy-L-proline (C4LHyp) and trans-4-hydroxy-L-proline (T4LHyp) are applied to build the chiral intermediates in pharmaceutical synthesis. Through comparisons chemical synthesis with biological catalysis to form hydroxy amino acids, enantioselective biocatalysts will undoubtedly be used in the synthesis of chiral pharmaceuticals. Mononuclear non-heme Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) that use an Fe(IV) complex intermediate to active diverse oxidative transformations with key biological roles. Studies identifying the important intermediates in catalysis and the proposed mechanisms are explained. In summary, we describe the physiological properties and synthesis regarding hydroxy amino acids, particularly 4-HIL and hydroxyproline. And the proposed catalysis mechanisms of Fe/αKGDs are also explained, while we also discussed the applications of hydroxy amino acids in fundamental research and industrial.

Potential Antileishmanial Activity of a Triazole‐Based Hybrid Peptide against Leishmania major

AbstractThe dipeptides 1–4 with a triazole containing synthetic amino acid and Leu/ Phe/ Val/ Gly have been designed and synthesized as a potent antileishmanial agent. The 1‐(2‐Amino‐phenyl)‐1H‐[1, 2,3] triazole‐4‐carboxylic acid was synthesized using click chemistry approach. From X‐ray crystallography, the peptide 1 containing a Leu residue at C‐terminus adopts kink like structure but there is no intramolecular hydrogen bond. So, the hydrogen bonding sites are free and allowed for intermolecular interaction with the guests. The phenylalanine analogue 2 where the phenyl and triazole rings are perpendicular to each other also form supramolecular column‐like structure through π‐π stacking interaction. The incorporation of a triazole containing synthetic amino acid at the N‐terminus enhances its anti‐parasites activity. The properties such as lipophilicity and cytotoxicity, are relevant factors for the design of new antileishmanial agent. IC50 value were calculated for dipeptides 1–4 using normal growth inhibition strategy. For dipeptide 1, IC50 value is 11μg/ml and the Phe containing dipeptide 2 exhibits IC50 21.17μg/ml on Leishmania major promastigotes. But the Val and Gly containing dipeptides 3 and 4 have IC50 values 138.23 μg/ml and 21.75 μg/ml respectively. The higher lipophilicity of dipeptide 1 is likely to improve the chances of reaching this intracellular parasite. The experimental result show that the triazole‐based dipeptide 1 is promising as antileishmanial agent.

Dynamic flux balance analysis for microbial conversion of glycerol into 1,3-propanediol by Klebsiella pneumoniae.

To investigate the relationship between the yield of 1,3-propanediol (1,3-PD) and the flux variation in metabolic pathways of Klebsiella pneumoniae, an optimized calculation method was constructed on basis of dynamic flux balance analysis by combining genome-scale flux balance analysis with a kinetic model of extracellular metabolites. Through optimizing calculations, a more completely expanded metabolic pathway was obtained, which includes the previously reported metabolic pathway and additional three pathways or site: a pentose phosphate pathway (PPP) elicited at the dihydroxyacetone (DHA) node to provide more reducing equivalents; a branch of synthetic amino acids at the 3-phosphoglycerate (3PG) node; and the α-ketoglutarate site in the tricarboxylic acid (TCA) cycle leading to anabolic pathways for glutamate and other amino acids. On this basis, the relationships between the dynamic flux distribution of the important nodes in the metabolic pathway and the yield of 1,3-propanediol were analyzed. First, dynamic flux change from DHA to the PPP is positively correlated with the yield. Second, variation in flux in the TCA cycle is also positively correlated with the yield of 1,3-propanediol. In addition, the influence of the feedback loop formed by the cofactor tetrahydrofolate on the flux change of TCA in the amino acid anabolic pathway was examined. These results are of important reference value and have guiding significance for the extension of the glycerol metabolism pathway in K. pneumoniae, the rational transformation of genetic engineering in bacteria, and the optimization of metabolic pathways for industrial production.

A biophysically-defined hyaluronic acid-based compound accelerates migration and stimulates the production of keratinocyte-derived neuromodulators

ABSTRACTHyaluronic acid (HA) preparations are widely used in clinical practice and recent data suggest that commercially available HA-based compounds promote ulcer re-epithelialization and induce pain relief. However, the pathophysiological basis of these effects remains poorly understood. In the present study, we investigated the biophysical, biomolecular and functional properties of a HA preparation combined with a pool of collagen precursor synthetic aminoacids, namely l-proline, l-leucine, l-lysine and glycine (Aminogam®). Hydrodynamic characterization of Aminogam® by size exclusion chromatography-triple detector array (SEC-TDA) revealed an average molecular weight in the range of 700–1700 kDa. Rheological measurements of the 1700kDa Mw lot showed a pseoudoplastic behaviour with a zero-shear viscosity (η0) equal to 90 ± 9 Pa∙s at 25°C and 55 ± 6 Pa∙s at 37°C. Automated time-lapse videomicroscopy studies in a fibroblast-free system demonstrated that 1% (v/v) Aminogam® significantly reduced the healing time of wounded keratinocyte monolayers. In AKGOS assays, Aminogam® stimulated cellular locomotion (chemokinesis) and directional migration (chemotaxis) of keratinocytes. Analysis of microarray data suggested that keratinocytes had a functional neuroendocrine machinery, and this was confirmed by testing the secretion of six neuroactive molecules by ELISA, namely α-MSH, β-endorphins, melatonin, substance P, cortisol, and neurotensin. Interestingly, Aminogam® regulated the production of several neuropeptides, including β-endorphins. In conclusion, our data shed light on the epithelial-dependent mechanisms that underlie the efficacy of Aminogam®, particularly in reference to wound healing and nociception.

Digestible methionine+cysteine in the diet of commercial layers and its influence on the performance, quality, and amino acid profile of eggs and economic evaluation.

This work aimed at evaluating the effects of 4 digestible Met+Cys levels on the diet of commercial layers and their influence on the productive performance, quality, and amino acid profile of eggs and economic viability of the activity. A total of 576 white Lohmann LSL-Lite layers was distributed into 6 replicates of 24 birds for each diet. The experimental design was completely randomized, with 4 treatments defined by levels evaluated in the feed (0.465, 0.540, 0.581, and 0.647%). The productive performance was measured for 30 weeks. The quality (34 and 50 wk old) and the amino acid profile of eggs (43 wk old) also were evaluated. A linear positive response was observed at higher Met+Cys levels for feed intake, number of eggs per housed bird, and digestible Met+Cys intake. Egg production, egg weight, egg mass, feed efficiency, and weight gain had their optimal values determined by the quadratic regression model at 0.638, 0.654, 0.647, 0.644, and 0.613% digestible Met+Cys, respectively. In the 34th wk, eggshell thickness decreased linearly at higher Met+Cys levels. In the 50th week, the optimal levels detected for eggshell thickness and percentage were 0.571 and 0.570% digestible Met+Cys, respectively. The percentages of proteins, branched-chain amino acids (leucine, isoleucine, and valine), histidine, and proline in eggs (albumen+yolk) showed a linear negative response in function of higher Met+Cys levels. Higher digestible Met+Cys levels (>0.630%) led to a good performance of layers, while lower Met+Cys levels improved the eggshell quality of layers in peak production. Optimal Met+Cys levels may change according to the price of the synthetic amino acid.

Reduction potential of odor gas emission from swine manure compost by using a low-protein diet supplemented with synthetic amino acid～Evaluation of Environmentally Friendly Diets : Productivity and environmental preservation～

悪臭抑制型飼料と期待されるアミノ酸バランス改善飼料の堆肥化処理過程における臭気抑制効果を総合的に検証した．慣行飼料に比べて生産物品質にも生産コストにも影響せず，環境負荷を軽減した臭気対策として提案できる．バランス改善飼料区では，堆積型堆肥化処理時のアンモニアの低減効果が認められ，茨城県では飼料用米と豆腐粕を原料とした発酵飼料を添加したオリジナルバランス飼料，山梨県ではバランス改善飼料をベースにブドウ粕由来の乳酸菌を添加することによる悪臭低減効果が検討された．

141 Effect of Low Protein Diets with or without Supplemented Synthetic Amino Acids on Growth Performance of Nursery Pigs.

141 Effect of Low Protein Diets with or without Supplemented Synthetic Amino Acids on Growth Performance of Nursery Pigs.

Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.

The Evolving Mechanisms of Action of Glatiramer Acetate.

Glatiramer acetate (GA) is a synthetic amino acid copolymer that is approved for treatment of relapsing remitting multiple sclerosis (RRMS) and clinically isolated syndrome (CIS). GA reduces multiple sclerosis (MS) disease activity and has shown comparable efficacy with high-dose interferon-β. The mechanism of action (MOA) of GA has long been an enigma. Originally, it was recognized that GA treatment promoted expansion of GA-reactive T-helper 2 and regulatory T cells, and induced the release of neurotrophic factors. However, GA treatment influences both innate and adaptive immune compartments, and it is now recognized that antigen-presenting cells (APCs) are the initial cellular targets for GA. The anti-inflammatory (M2) APCs induced following treatment with GA are responsible for the induction of anti-inflammatory T cells that contribute to its therapeutic benefit. Here, we review studies that have shaped our current understanding of the MOA of GA.

Total Synthesis of Ecumicin.

The first total synthesis of the potent anti-mycobacterial cyclic depsipeptide natural product ecumicin is described. Synthesis was achieved via a solid-phase strategy, incorporating the synthetic non-proteinogenic amino acids N-methyl-4-methoxy-l-tryptophan and threo-β-hydroxy-l-phenylalanine into the growing linear peptide chain. The synthesis employed key on-resin esterification and dimethylation steps as well as a final macrolactamization between the unusual N-methyl-4-methoxy-l-tryptophan unit and a bulky N-methyl-l-valine residue. The synthetic natural product possessed potent antimycobacterial activity against the virulent H37Rv strain of Mycobacterium tuberculosis (MIC90 = 312 nM).

Orthogonality of a Synthetic Pyrrolysine tRNA in the Xenopus Oocyte and its use to Encode Unnatural Amino Acids into Ion Channels

The power of the chemical misacylation method of nonsense suppression stems from the ability to attach a wide range of synthetic amino acids to tRNA without the need to evolve specific aminacyl-tRNA synthetases. This method has been powerfully paired with protein expression in the Xenopus oocyte, wherein injection of the target RNA and tRNA is facile. However, the fidelity of the method depends on the orthogonality of the tRNA species being used to deliver the amino acid; the tRNA must be accepted by the ribosomal machinery of the cell but ignored by endogenous aminacyl-tRNA synthetases that may edit the tRNA with a natural amino acid. Tetrahymena thermophila -based tRNAs are orthogonal but have been shown to be re-edited under some conditions. To improve the method and expand its uses, we asked whether the Pyrrolysine tRNA from Barkeri fusaro (PylT), which has already been shown to be highly orthogonal in mammalian cells, could be used in nonsense suppression experiments in the Xenopus oocyte. Simultaneously, we also asked whether we could use fully synthetic (as opposed to in vitro transcribed) tRNA for this application. Functional and biochemical experiments indicate that PylT is orthogonal in the oocyte, and that it can be used to deliver diverse amino acids into multiple targets (including voltage-gated sodium channels, voltage gated potassium channels and sodium transporters) to achieve high expression of engineered proteins with limited evidence of ‘bleedthrough’ of the introduced TAG codon within the target cRNA. The demonstration that fully synthetic tRNA is useful in nonsense suppression eliminates the need for costly and tedious in vitro transcription and gel filtration in the manufacture of aminacyl-tRNA pellets, and should expand access to this method.