Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

2006 ◽  
Vol 1 (6) ◽  
pp. 2957-2962 ◽  
Author(s):  
Nobumasa Hino ◽  
Akiko Hayashi ◽  
Kensaku Sakamoto ◽  
Shigeyuki Yokoyama
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Mammalian Cells ◽  
Site Specific ◽  
Natural Amino Acids ◽  
Expanded Genetic Code

scholarly journals Protein evolution with an expanded genetic code

2008 ◽  
Vol 105 (46) ◽  
pp. 17688-17693 ◽  
Author(s):  
Chang C. Liu ◽  
Antha V. Mack ◽  
Meng-Lin Tsao ◽  
Jeremy H. Mills ◽  
Hyun Soo Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Unnatural Amino Acids ◽  
Selective Advantage ◽  
Display System ◽  
Antibody Library ◽  
Evolution Of Proteins ◽  
Expanded Genetic Code ◽  
Selection Of

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

scholarly journals Exploring the potential impact of an expanded genetic code on protein function

2015 ◽  
Vol 112 (22) ◽  
pp. 6961-6966 ◽  
Author(s):  
Han Xiao ◽  
Fariborz Nasertorabi ◽  
Sei-hyun Choi ◽  
Gye Won Han ◽  
Sean A. Reed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Conformational Changes ◽  
Protein Function ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
Functional Changes ◽  
Living Organisms ◽  
Expanded Genetic Code

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells

2003 ◽  
Vol 10 (6) ◽  
pp. 573-580 ◽  
Author(s):  
Sarah L Monahan ◽  
Henry A Lester ◽  
Dennis A Dougherty
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Site Specific

scholarly journals High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

scholarly journals Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

2019 ◽  
Author(s):  
Junetha Syed ◽  
Saravanan Palani ◽  
Scott T. Clarke ◽  
Zainab Asad ◽  
Andrew R. Bottrill ◽  
...  
Keyword(s):  
Genetic Code ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Glutathione S Transferase ◽  
Base Pairs ◽  
Site Specific ◽  
Green Fluorescent ◽  
Natural Amino Acids ◽  
Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

Formylglycine-generating enzymes for site-specific bioconjugation

2019 ◽  
Vol 400 (3) ◽  
pp. 289-297 ◽  
Author(s):  
Tobias Krüger ◽  
Thomas Dierks ◽  
Norbert Sewald
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Proteins ◽  
The State ◽  
Protein Scaffolds ◽  
Site Specific ◽  
Catalytic Mechanisms ◽  
Consensus Motifs ◽  
State Of Research ◽  
Natural Amino Acids

Abstract Site-specific bioconjugation strategies offer many possibilities for directed protein modifications. Among the various enzyme-based conjugation protocols, formylglycine-generating enzymes allow to posttranslationally introduce the amino acid Cα-formylglycine (FGly) into recombinant proteins, starting from cysteine or serine residues within distinct consensus motifs. The aldehyde-bearing FGly-residue displays orthogonal reactivity to all other natural amino acids and can, therefore, be used for site-specific labeling reactions on protein scaffolds. In this review, the state of research on catalytic mechanisms and consensus motifs of different formylglycine-generating enzymes, as well as labeling strategies and applications of FGly-based bioconjugations are presented.

scholarly journals Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence

2015 ◽  
Vol 6 (2) ◽  
pp. 1159-1166 ◽  
Author(s):  
Samuel C. Reddington ◽  
Amy J. Baldwin ◽  
Rebecca Thompson ◽  
Andrea Brancale ◽  
Eric M. Tippmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Target Protein ◽  
Natural Amino Acids

Genetic code reprogramming allows proteins to sample new chemistry through targeted introduction of non-natural amino acids. By combining with random codon replacement, residues traditionally overlooked can be identified as instilling new properties on a target protein.

scholarly journals Evolving Bacterial Fitness with an Expanded Genetic Code

2017 ◽  
Author(s):  
Drew S. Tack ◽  
Austin C. Cole ◽  
R. Shroff ◽  
B.R. Morrow ◽  
Andrew D. Ellington
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Bacterial Fitness ◽  
Genetic Code Expansion ◽  
Encoding Strategy ◽  
Translation Systems ◽  
Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

scholarly journals Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

2021 ◽  
Vol 22 (20) ◽  
pp. 11194
Author(s):  
Nikolaj G. Koch ◽  
Peter Goettig ◽  
Juri Rappsilber ◽  
Nediljko Budisa
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Cell Biology ◽  
Trna Synthetase ◽  
Side Chains ◽  
Site Specific ◽  
Biomaterial Science ◽  
Key Residues ◽  
Small Side

Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology. The diversification of the repertoire of the genetic code has been achieved for amino acids with long and/or bulky side chains equipped with various bioorthogonal tags and useful spectral probes. Although ncAAs with relatively small side chains and similar properties are of great interest to biophysics, cell biology, and biomaterial science, they can rarely be incorporated into proteins. To address this gap, we report the engineering of PylRS variants capable of incorporating an entire library of aliphatic “small-tag” ncAAs. In particular, we performed mutational studies of a specific PylRS, designed to incorporate the shortest non-bulky ncAA (S-allyl-l-cysteine) possible to date and based on this knowledge incorporated aliphatic ncAA derivatives. In this way, we have not only increased the number of translationally active “small-tag” ncAAs, but also determined key residues responsible for maintaining orthogonality, while engineering the PylRS for these interesting substrates. Based on the known plasticity of PylRS toward different substrates, our approach further expands the reassignment capacities of this enzyme toward aliphatic amino acids with smaller side chains endowed with valuable functionalities.

Sign in / Sign up

Export Citation Format

Share Document