A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

A biological boronate: An orthogonal tRNA/aminoacyl‐tRNA synthetase pair has been evolved for the genetic incorporation of a boronic acid into proteins. This amino acid has been used to purify proteins in a one‐step scarless purification procedure as well as for the site‐specific labeling of proteins using various boronic acid chemistries.

An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in E. coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biological processes with light, including enzyme, receptor, and ion channel activity.

Activating proteins with light: A photocaged tyrosine was genetically encoded in E. coli in response to the amber codon TAG. Substitution of Tyr 503 in the active site of β‐galactosidase allowed photoactivation of this enzyme in vitro or directly in bacteria with 360‐nm light. This method should allow photoregulation of the activity of a variety of biological processes including transcription, signal transduction, and cellular trafficking.

Regions of the M. jannaschii tyrosyl tRNA CUA thought to interact with elongation factor Tu were randomized, and the resulting tRNA libraries were subjected to in vitro evolution. The tRNAs identified resulted in significantly improved unnatural amino acid-containing protein yields. In some cases, the degree of improvement varied in an amino acid-dependent manner. KeywordstRNA; EF-Tu; evolution; amber suppression; unnatural amino acids tRNAs have evolved to act as highly efficient amino acid carriers and activators during each stage of protein synthesis. Each tRNA must be selectively charged by its cognate aminoacyltRNA synthetase (aaRS); the resulting aminoacyl-tRNA must be efficiently bound by * Fax: (+1) 858-784-9440, schultz@scripps.edu. + These authors contributed equally to this work. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript elongation factor Tu (EF-Tu) for transport to the ribosome; after binding to the ribosomal A site, the aminoacyl-tRNA must function efficiently in translation as a substrate for the peptidyl transferase; and finally the tRNA bearing the growing peptide chain must be translocated to the P site, undergo another acyl transfer reaction, and be released from the ribosome. We and others have used orthogonal tRNA/aaRS pairs for the site-specific incorporation of nearly 50 unnatural amino acids in E. coli, S. cerevisiae, and mammalian cells in response to unique nonsense and frameshift codons.1 An engineered M. jannaschii amber suppressor tyrosyl-tRNA/tRNA-synthetase ( /MjYRS) pair has been the most extensively used system for the evolution of aaRS variants that incorporate unnatural amino acids in E. coli. Although unnatural amino acids are typically incorporated into proteins with good efficiency and excellent fidelity, further system optimization resulting in increased protein yields is highly desirable.Because is derived from an archaeal tRNA and therefore significantly differs in sequence from E. coli tRNAs, it may not function optimally with the E. coli translational machinery. Furthermore, in vitro binding studies have shown that while correctly acylated tRNAs bind EF-Tu with near uniform affinity, tRNAs bearing non-cognate amino acids show a broad range of affinities for EF-Tu2, indicating that the tRNA body and the esterified amino acid make compensatory contributions to EF-Tu binding. A number of genetic, biochemical, and structural studies have implicated specific residues within the tRNA acceptor stem and T stem as being important for EF-Tu binding.3 tRNA misacylation has also recently been shown to perturb binding to the ribosomal A site.4 Therefore it is likely that tRNAs acylated with noncognate unnatural amino acids adversely impact the efficiency of protein synthesis due to non-optimal interactions with EF-Tu and/or the ribosome.We have used in vitro evolution to optimize the sequence of with MjYRS and a panel of six evolved unnatural amino acid incorporating MjYRS variants, and have identified several unique tRNA seq...

Angewandte Evolution: Bereiche der M.‐jannaschii‐Tyrosyl‐tRNACUA, von denen man annimmt, dass sie mit dem Elongationsfaktor Tu wechselwirken, wurden randomisiert und die erhaltenen tRNA‐Bibliotheken der In‐vitro‐Evolution unterworfen. Die dabei identifizierten tRNAs lieferten Proteine mit nichtnatürlichen Aminosäuren in deutlich höheren Ausbeuten. Manchmal hing das Ausmaß der Verbesserung von der Aminosäure ab.

Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins, mAbs, 6:3, 671-678,

Ein Shuttle‐System zur Verwendung von in E. coli selektierten Aminoacyl‐tRNA‐Synthetasen (aaRSs) für den Einbau von nichtnatürlichen Aminosäuren in Säugerzellen wurde entwickelt. Eine in E. coli selektierte Mutante der Pyrrolysyl‐tRNA‐Synthetase (PylRS) aminoacyliert selektiv eine Nonsense‐Suppressor‐tRNA mit einem photoaktivierbaren Lysinderivat. Durch Übertragung dieses orthogonalen tRNA‐aaRS‐Paares in Säugerzellen gelang es, diese nichtnatürliche Aminosäure selektiv in Proteine einzubauen.