Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe

Abstract The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms. In nearly all cases, the amber codon is used as a sense codon and an orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair is used to generate amber suppressing tRNAs charged with the UAA. In this work, we have developed tools to incorporate the cross-linking amino acid azido-phenylalanine (AzF) through the use of bacterial tRNATyr and a modified version of TyrRS, AzFRS, in Schizosaccharomyces pombe, which is an attractive model organism for the study of cell behavior and function. We have incorporated AzF into three different proteins. We show that the majority of AzF is modified to amino-phenyl alanine, but protein cross-linking was still observed. These studies set the stage for exploitation of this new technology for the analysis of S. pombe proteins.

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Optimization of the posttranslational click modification of proteins

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc2011103 ◽

2011 ◽

Vol 76 (9) ◽

pp. 1089-1101

Author(s):

Milan Vrabel ◽

Emine Kaya ◽

Stefan Prill ◽

Veronika Ehmke ◽

Thomas Carell

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Fluorescent Protein ◽

Click Reaction ◽

Yellow Fluorescent Protein ◽

Trna Synthetase ◽

Site Specific ◽

Specific Manner ◽

Modified Proteins ◽

A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

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

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03417-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1718-1725 ◽

Cited By ~ 16

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.

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Site-specific modification of albumin by free radicals. Reaction with copper(II) and ascorbate

Biochemical Journal ◽

10.1042/bj2360397 ◽

1986 ◽

Vol 236 (2) ◽

pp. 397-400 ◽

Cited By ~ 148

Author(s):

G Marx ◽

M Chevion

Keyword(s):

Gel Electrophoresis ◽

Chelating Agents ◽

Polyacrylamide Gel Electrophoresis ◽

Cross Linking ◽

Oh Radicals ◽

Site Specific ◽

Metal Chelating ◽

Specific Manner ◽

A Site ◽

New Protein

Exposure of albumin to Cu(II) (10-100 microM) and ascorbate (0.1-2 mM) results in extensive molecular modifications, indicated by decreased fluorescence and chain breaks. The rate of utilization of molecular oxygen and ascorbate as a function of Cu(II) concentration is non-linear at copper/albumin ratios of greater than 1. It appears that Cu(II) bound to the tightest albumin-binding site is less available to the ascorbate than the more loosely bound cation. SDS/polyacrylamide-gel electrophoresis reveals new protein bands corresponding to 50, 47, 22, 18 and 3 kDa. For such a cleavage pattern, relatively few (approximately 3) and rather specific chain breaks occurred. Repeated addition of portions of ascorbate to the albumin/Cu(II) mixture results in increased intensity of the new bands. The absence of Cu(II) or the presence of metal chelating agents is inhibitory. There was no evidence of intermolecular cross-linking or of the formation of insoluble, albumin-derived, material. A mechanism is proposed wherein the loosely bound Cu(II) participates in a Fenton-type reaction. This generates OH. radicals, which rapidly inter-react with the protein and modify it in a ‘site-specific’ manner.

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Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.94.19.10092 ◽

1997 ◽

Vol 94 (19) ◽

pp. 10092-10097 ◽

Cited By ~ 116

Author(s):

D. R. Liu ◽

T. J. Magliery ◽

M. Pastrnak ◽

P. G. Schultz

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Site Specific

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Faculty Opinions recommendation of Site-specific protein immobilization using unnatural amino acids.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.723878764.793522625 ◽

2016 ◽

Author(s):

Morten Meldal ◽

Sanne Schoffelen

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Protein Immobilization ◽

Specific Protein ◽

Site Specific

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Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37331-3 ◽

1994 ◽

Vol 269 (10) ◽

pp. 7610-7616

Author(s):

C.T. Kuan ◽

Q.C. Wang ◽

I. Pastan

Keyword(s):

Polyethylene Glycol ◽

Cysteine Residues ◽

Exotoxin A ◽

Pseudomonas Exotoxin ◽

Site Specific ◽

Pseudomonas Exotoxin A ◽

Specific Manner ◽

A Site

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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 ◽

10.1007/s00726-020-02927-z ◽

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.

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