Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, *

The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.

BackgroundCarbon dioxide (CO2) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector Anopheles gambiae sensu stricto is attracted to human volatiles augmented by CO2. This study investigated whether CO2, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO2 derived from fermenting yeast (yeast-produced CO2).MethodsTrapping experiments were conducted in the laboratory, semi-field and field, with An. gambiae s.s. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO2. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO2 on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ2-test, the field data by GLM. In addition, CO2 concentrations produced by yeast-sugar solutions were measured over time.ResultsTraps baited with yeast-produced CO2 caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO2, both in the laboratory and semi-field. Adding yeast-produced CO2 to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO2 + human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. Anopheles gambiae s.s. was not caught during the field trials. However, traps baited with yeast-produced CO2 caught similar numbers of Anopheles arabiensis as traps baited with industrial CO2. Addition of human odour increased trap catches.ConclusionsYeast-produced CO2 can effectively replace industrial CO2 for sampling of An. gambiae s.s.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.