Tailoring the Substrate Specificity of Yeast Phenylalanyl-tRNA Synthetase toward a Phenylalanine Analog Using Multiple-Site-Specific Incorporation

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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Valyl-tRNA, Isoleucyl-tRNA and Tyrosyl-tRNA Synthetase from Baker's Yeast. Substrate Specificity with Regard to ATP Analogs and Mechanism of the Aminoacylation Reaction

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1976.tb10574.x ◽

1976 ◽

Vol 66 (3) ◽

pp. 493-497 ◽

Cited By ~ 25

Author(s):

Wolfgang FREIST ◽

Friedrich HAAR ◽

Heinz FAULHAMMER ◽

Friedrich CRAMER

Keyword(s):

Substrate Specificity ◽

Trna Synthetase ◽

Baker’S Yeast ◽

Baker's Yeast ◽

Atp Analogs

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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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Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain through Site-Specific Mutations†

Biochemistry ◽

10.1021/bi015864r ◽

2001 ◽

Vol 40 (51) ◽

pp. 15464-15470 ◽

Cited By ~ 135

Author(s):

Christopher D. Reeves ◽

Sumati Murli ◽

Gary W. Ashley ◽

Misty Piagentini ◽

C. Richard Hutchinson ◽

...

Keyword(s):

Substrate Specificity ◽

Polyketide Synthase ◽

Site Specific ◽

Modular Polyketide Synthase

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Substrate Specificity of the Oncoprotein v-Fps: Site-Specific Mutagenesis of the Putative P+1 Pocket†

Biochemistry ◽

10.1021/bi992096j ◽

2000 ◽

Vol 39 (1) ◽

pp. 255-262 ◽

Cited By ~ 5

Author(s):

Laurel Konkol ◽

T. John Hirai ◽

Joseph A. Adams

Keyword(s):

Substrate Specificity ◽

Site Specific

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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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