Exploiting redundancy of the genetic code for site-selective unnatural amino acids incorporation in vitro

2016 ◽  
Author(s):  
Zhenling Cui
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Site Selective

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

The Enzymatic Degradation of Angiotensin II Analogues by proteolytic enzyme in vitro

1972 ◽  
Vol 50 (2) ◽  
pp. 113-118 ◽  
Author(s):  
M. C. Carrara ◽  
D. Regoli ◽  
W. K. Park
Keyword(s):  
Amino Acids ◽  
Angiotensin Ii ◽  
Unnatural Amino Acids ◽  
Enzymatic Degradation ◽  
Leucine Aminopeptidase ◽  
Receptor Interaction ◽  
Angiotensin I ◽  
Peptide Receptor ◽  
Angiotensin Ii Analogues

Angiotensin II (ATII), angiotensin I (ATI), and Acpc analogues of ATII, 8-Achc-ATII, 8-D-Phe-ATII, and 8-Ala-ATII, were incubated in vitro with carboxypeptidase, chymotrypsin, and leucine-aminopeptidase in order to study the influence of unnatural amino acids (Acpc, Achc, and D-Phe) and of L-Ala on the activity of peptidases.Fragments occurring during the breakdown of peptides were demonstrated by paper chromatography in an ascending system.ATII and ATI are rapidly inactivated by carboxypeptidase and chymotrypsin, while the degradation by leucine-aminopeptidase is slower.Substitution of L-Phe with Acpc, Achc, D-Phe, or L-Ala in position 8 prevents the degradation by carboxypeptidase. Chymotrypsin degrades 3-Acpc-ATII and 5-Acpc-ATII but not 4-Acpc-ATII. The action of leucine-aminopeptidase is not influenced by substituting Acpc to each one of the first five amino acids composing the molecule of angiotensin.The possible implications of these findings for the peptide-receptor interaction is discussed.

scholarly journals Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

2010 ◽  
Vol 132 (42) ◽  
pp. 14819-14824 ◽  
Author(s):  
Susan M. Hancock ◽  
Rajendra Uprety ◽  
Alexander Deiters ◽  
Jason W. Chin
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase

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.

Synthesis and in vitro antitumor activity of new octapeptide analogs of somatostatin containing unnatural amino acids

Amino Acids ◽  
2015 ◽  
Vol 47 (5) ◽  
pp. 1007-1013 ◽  
Author(s):  
Svetlana Ts. Staykova ◽  
Diana W. Wesselinova ◽  
Lyubomir T. Vezenkov ◽  
Emilia D. Naydenova
Keyword(s):  
Amino Acids ◽  
Antitumor Activity ◽  
Unnatural Amino Acids

scholarly journals Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids

1996 ◽  
Vol 3 (12) ◽  
pp. 1033-1038 ◽  
Author(s):  
Sharon T. Cload ◽  
David R. Liu ◽  
Wayne A. Froland ◽  
Peter G. Schultz
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
In Vitro Biosynthesis

scholarly journals Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

2019 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Michael Shaferman ◽  
Ben Zaad ◽  
Rambabu Dandela ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
High Efficiency ◽  
Living Cells ◽  
Trna Synthetase ◽  
Complex Nature ◽  
Reporter Protein ◽  
A Cell ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

scholarly journals Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

2021 ◽  
Author(s):  
Xuesheng Wu ◽  
Zhetao Zheng ◽  
Hongmin Chen ◽  
Haishuang Lin ◽  
Yuelin Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Influenza A ◽  
Antiviral Agent ◽  
Unnatural Amino Acid ◽  
Drug Resistant ◽  
Genetic Code Expansion

AbstractThe frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

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