scholarly journals Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1

2014 ◽  
Vol 136 (44) ◽  
pp. 15577-15583 ◽  
Author(s):  
Wolfgang H. Schmied ◽  
Simon J. Elsässer ◽  
Chayasith Uttamapinant ◽  
Jason W. Chin
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells

2009 ◽  
Vol 5 (9) ◽  
pp. 931 ◽  
Author(s):  
Jeffrey K. Takimoto ◽  
Katrina L. Adams ◽  
Zheng Xiang ◽  
Lei Wang
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

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.

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells

Science ◽  
1995 ◽  
Vol 268 (5209) ◽  
pp. 439-442 ◽  
Author(s):  
M. Nowak ◽  
P. Kearney ◽  
Sampson ◽  
M. Saks ◽  
C. Labarca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Receptor Binding ◽  
Nicotinic Receptor ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Receptor Binding Site ◽  
Intact Cells ◽  
Acid Incorporation

Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates

2014 ◽  
Vol 31 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Prashanta Shrestha ◽  
Mark Thomas Smith ◽  
Bradley Charles Bundy
Keyword(s):  
Amino Acid ◽  
Alternative Energy ◽  
Energy Systems ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Expression Templates ◽  
Linear Expression

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 Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding

ChemBioChem ◽  
2016 ◽  
Vol 17 (11) ◽  
pp. 981-984 ◽  
Author(s):  
Taehyung C. Lee ◽  
Minjin Kang ◽  
Chan Hyuk Kim ◽  
Peter G. Schultz ◽  
Eli Chapman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Click Chemistry ◽  
Single Molecule ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Single Molecule Fret ◽  
Acid Incorporation

Efficient unnatural amino acid incorporation

2016 ◽  
Vol 13 (2) ◽  
pp. 116-116
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation
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