Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code Han Xiao1 and Peter G. Schultz1,2 1Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037 2California Institute for Biomedical Research, La Jolla, California 92037 Correspondence: [email protected] The ability to site-specifically incorporate noncanonical amino acids (ncAAs) with novel structures into proteins in living cells affords a powerful tool to investigate and manipulate protein structure and function. More than 200 ncAAs with diverse biological, chemical, and physical properties have been genetically encoded in response to nonsense or frameshift codons in both prokaryotic and eukaryotic organisms with high fidelity and efficiency. In this review, recent advances in the technology and its application to problems in protein bio- chemistry, cellular biology, and medicine are highlighted. ith the rare exceptions of pyrrolysine and tase (aaRS)/transfer RNA (tRNA) pair that is Wselenocysteine, the genetic codes of all specific for the ncAA but does not cross-react known organisms consist of the same 20 canon- with endogenous host aaRSs, tRNAs, or amino ical amino acids. However, additional function- acids (Fig. 1A) (Wang et al. 2006b; Liu and al groups, including organic and inorganic Schultz 2010). Using this system, over 200 struc- cofactors and posttranslational modifications turally distinct ncAAs have been genetically (PTMs), are required for many of the functions encoded in both prokaryotic and eukaryotic performed by proteins. This observation sug- organisms. These ncAAs include spectroscopic gests that an expanded genetic code might allow probes, metal ion chelators, photo-affinity one to either rationally design or evolve proteins probes and photocaged amino acids, posttrans- with new or enhanced physical, chemical, and lational modifications, and amino acids with biological properties. To this end, methodology orthogonal chemical activity for the site-specific has been developed to exploit the endogenous modification of proteins (Wanget al. 2006b; Liu protein biosynthetic machinery to site-specif- and Schultz 2010; Wan et al. 2014). They have ically incorporate noncanonical amino acids been used as probes of protein structure and (ncAAs) into proteins in living organisms with function, both in vitro and in vivo, and in the high translational fidelity and efficiency (up to rational design of proteins with neworenhanced 5g/L on a commercial scale). The desired ncAA biological or pharmacological activities. Most is encoded by a nonsense or frameshift codon recently, in vitro evolution experiments have be- using an orthogonal aminoacyl-tRNA synthe- gun to show that additional amino acid building Editors: Daniel G. Gibson, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter Additional Perspectives on Synthetic Biology available at www.cshperspectives.org Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a023945 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023945 1 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press H. Xiao and P.G. Schultz blocks can lead to novel protein structures and amino acid side chains; however, the efficiencies activities. Herein, we review a number of recent with which the ncAAs can be incorporated at a advances in the field, both in the development given site in the proteome vary—ranging from and application of this technology. milligrams to 5þ g/L of mutant protein, most likely because of the differing degrees to which the aaRS/tRNA pair is optimized (of course, for GENETICALLY ENCODING ncAAs any given protein, efficiency is also affected by Evolved Methanococcus jannaschii tyrosyl aaRS/ the mutation site). Platform vectors, for exam- tRNA pairs and Methanosarcina barkeri and ple, pEvol and pUltra, have been established that mazei pyrrolysyl aaRS/tRNA pairs have enabled allow high-level protein expression of a desired the incorporation of a large number of ncAAs mutant protein in bacteria, and large-scale into proteins in Escherichia coli (Fig. 1B) (Wang fermentation (.10,000 L) has yielded mutant et al. 2006b; Liu and Schultz 2010; Wan et al. proteins on a 5 g/L scale (Young et al. 2010; 2014). To further expand the number and na- Chatterjee et al. 2013b). A current focus is the ture of genetically encoded ncAAs, aaRS/tRNA efficient incorporation of multiple identical or pairs from other archeal and eukaryotic organ- distinct ncAAs into the same protein, or the syn- isms have been recently developed, includ- thesis of an entirely “unnatural” biopolymer. ing Pyrococcus horikoshii lysyl aaRS/tRNA, One hypothesis is that the presence of endoge- P.horikoshii glutamyl aaRS/tRNA, Saccharomy- nous release factors leads to low overall incorpo- ces cerevisiae tryptophanyl aaRS/tRNA, hetero- ration efficiency for multiple ncAAs encoded geneous leucyl Mt-tRNA/Hs-aaRS, and proly by nonsense codons. Huang et al. (2010) first Af-tRNA/Ph-aaRS pairs (Anderson and Schultz showed that up to three ncAAs could be genet- 2003; Santoro et al. 2003; Anderson et al. 2004; ically incorporated into the same protein by Chatterjee et al. 2012, 2013d; Xiao et al. 2014). overexpression of the carboxy-terminal domain The structurally distinct active sites of these of ribosomal protein L11 to reduce release factor aaRSs allow one to encode chemically diverse 1 (RF1)-mediated termination of protein trans- A X Noncanonical Canonical amino acids amino acids Orthogonal Endogenous synthetase synthetase AMP+PPi AMP+PPi mRNA A-G- C-A- C-U- U-A-U-3′ U- U-U- A-C- ′5-U-A-A-C-A- A-U- C- Translation Proteineinin Figure 1. Figure continues on following page. 2 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023945 Downloaded from Cite this article as http://cshperspectives.cshlp.org/ O B H O N3 I Br O O N CH3 CH3 N3 O H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H Cold Spring Harb Perspect Biol (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) OH F B F OCF3 CF3 CN NO2 OH OH OH F OH OH OH N I NH2 NO2 F H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) onOctober1,2021-PublishedbyColdSpringHarborLaboratoryPress NN O O O O Br O N N O CH3 HN O HN HN O NH NH NH O O N3 N 2 H N CH3 O H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) 2016;8:a023945 H O N3 O O O O O O H H N NH CH O O OH HN O HN O 3 NH HN 2 N HN O HN O HN O HN 3 H 2 N SH N N H SH N O H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) O NO2 OMe O N O O N OMe O 2 HN O HN O O NH O O2N O2N N H O O N HN O S O O NO S 2 O O H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H Expanding the Genetic Code (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) O N N O O O O Br CH H C CH HN O CH O HN HN HN 3 3 N 3 HN HN 3 H OH N F OH OSO - S 3 O O O H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H H2N CO2H (53) (54) (55) (56) (57) (58) (59) (60) (61) (62) (63) Figure 1. Expanding the genetic code. (A) The site-specific incorporation of noncanonical amino acids (ncAAs) using an orthogonal aminoacyl-tRNA synthetase (aaRS)/transfer RNA (tRNA) pair. (B) ncAAs described in this review. mRNA, Messenger RNA. 3 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press H. Xiao and P.G. Schultz lation. Using an RF1 knockout strain, Johnson with bio-orthogonal chemical reactivities (e.g., et al. (2011) showed that up to 10 p-acetylphenyl- azido, alkynyl, and tetrazinyl), fluorescent alanines (pAcF; Fig. 1B1) could be incorporated probes, PTMs, and photocaged amino acids into the same protein, albeit in relatively low yield (Fig. 1B) (Wang et al. 2006b; Liu and Schultz (Fig. 2A). Knockout of RF1 improves ncAA in- 2010; Wanet al. 2014). These orthogonal tRNA/ corporation in response to the amber codon; aaRS pairs are either evolved in yeast (for however, impaired growth rates resulting from E. coli–derived pairs) or bacteria (for archaea- RF1 deletion may limit the utility of this strain. derived pairs) and then transferred to vectors Recently, multiplex automated genome en- with appropriate promoters for transient ex- gineering (MAGE) and conjugative assembly pression in the host cell, typically, HEK293 or genome engineering (CAGE) were used to re- CHO cells. Yields of transiently expressed pro- place all of the TAG stop codons in the E. coli teins on the order of 5–20 mg/L are common, MG1655 genome with the TAA ochre nonsense and yields up to 1 g/L have been reported for codon (Fig. 2A) (Lajoie et al. 2013); subse- stable cell lines expressing mutant full-length quently, RF1 was knocked out to generate antibodies (Tian et al.