scholarly journals Expanding the Library and Substrate Diversity of the Pyrrolysyl-tRNA Synthetase to Incorporate Unnatural Amino Acids Containing Conjugated Rings

ChemBioChem ◽  
2013 ◽  
Vol 14 (16) ◽  
pp. 2100-2105 ◽  
Author(s):  
Vanessa K. Lacey ◽  
Gordon V. Louie ◽  
Joseph P. Noel ◽  
Lei Wang
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase

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.

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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 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 Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a Lactococcus lactis Trp Auxotroph

2016 ◽  
Vol 26 (4) ◽  
pp. 269-276
Author(s):  
Jinfeng Shao ◽  
Marcelo F.M. Marcondes ◽  
Vitor Oliveira ◽  
Jaap Broos
Keyword(s):  
Amino Acids ◽  
New Media ◽  
Lactococcus Lactis ◽  
Unnatural Amino Acids ◽  
Defined Medium ◽  
Trna Synthetase ◽  
Growth Media ◽  
Heterologous Proteins ◽  
Chemically Defined Media ◽  
Defined Media

Chemically defined media for growth of <i>Lactococcus lactis</i> strains contain about 50 components, making them laborious and expensive growth media. However, they are crucial for metabolism studies as well as for expression of heterologous proteins labeled with unnatural amino acids. In particular, the <i>L. lactis</i> Trp auxotroph PA1002, overexpressing the tryptophanyl tRNA synthetase enzyme of <i>L. lactis</i>, is very suitable for the biosynthetic incorporation of Trp analogs in proteins because of its most relaxed substrate specificity reported towards Trp analogs. Here we present two much simpler defined media for <i>L. lactis</i>, which consist of only 24 or 31 components, respectively, and with which the <i>L. lactis</i> Trp auxotroph shows similar growth characteristics as with a 50-component chemically defined medium. Importantly, the expression levels of two recombinant proteins used for evaluation were up to 2-3 times higher in these new media than in the 50-component medium, without affecting the Trp analog incorporation efficiency. Taken together, the simplest chemically defined media reported so far for <i>L. lactis</i> are presented. Since<i> L. lactis</i> also shows auxotrophy for Arg, His, Ile, Leu Val, and Met, our simplified media may also be useful for the biosynthetic incorporation of analogs of these five amino acids.

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.

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