Rapid Identification of Functional Pyrrolysyl-tRNA Synthetases via Fluorescence-Activated Cell Sorting

The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins.

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Directed Evolution of the Substrate Specificities of a Site-Specific Recombinase and an Aminoacyl-tRNA Synthetase Using Fluorescence-Activated Cell Sorting (FACS)

Directed Enzyme Evolution ◽

10.1385/1-59259-396-8:291 ◽

2003 ◽

pp. 291-312 ◽

Cited By ~ 1

Author(s):

Stephen W. Santoro ◽

Peter G. Schultz

Keyword(s):

Directed Evolution ◽

Cell Sorting ◽

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Fluorescence Activated Cell Sorting ◽

Site Specific ◽

Substrate Specificities ◽

A Site

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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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Fidelity of Phenylalanyl-tRNA Synthetase in Binding the Natural Amino Acids

The Journal of Physical Chemistry B ◽

10.1021/jp034607o ◽

2003 ◽

Vol 107 (41) ◽

pp. 11549-11557 ◽

Cited By ~ 10

Author(s):

Peter M. Kekenes-Huskey ◽

Nagarajan Vaidehi ◽

Wely B. Floriano ◽

William A. Goddard

Keyword(s):

Amino Acids ◽

Trna Synthetase ◽

Natural Amino Acids

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Co-assembly of helical β3-peptides: a self-assembled analogue of a statistical copolymer

Pure and Applied Chemistry ◽

10.1515/pac-2017-0709 ◽

2017 ◽

Vol 89 (12) ◽

pp. 1809-1816 ◽

Cited By ~ 2

Author(s):

Claire Buchanan ◽

Christopher J. Garvey ◽

Patrick Perlmutter ◽

Adam Mechler

Keyword(s):

Amino Acids ◽

Physical Properties ◽

Self Assembly ◽

Unnatural Amino Acids ◽

The Self ◽

Microscopy Imaging ◽

Wide Range ◽

Self Assembled ◽

Unnatural Peptides ◽

Natural Amino Acids

AbstractUnnatural peptide self-assembly offers the means to design hierarchical nanostructures of controlled geometries, chemical function and physical properties. N-acyl β3 peptides, where all residues are unnatural amino acids, are able to form helical fibrous structures by a head-to-tail assembly of helical monomers, extending the helix via a three point supramolecular hydrogen bonding motif. These helical nanorods were shown to be stable under a wide range of physical conditions, offering a self-assembled analogue of polymeric fibres. Hitherto the self-assembly has only been demonstrated between identical monomers; however the self-assembly motif is sequence-independent, offering the possibility of hetero-assembly of different peptide monomers. Here we present a proof of principle study of head-to-tail co-assembly of two different helical unnatural peptides Ac-β3[WELWEL] and Ac-β3[LIA], where the letters denote the β3 analogues of natural amino acids. By atomic force microscopy imaging it was demonstrated that the homo-assembly and co-assembly of these peptides yield characteristically different structures. Synchrotron small angle X-ray scattering experiments have confirmed the presence of the fibres in the solution and the averaged diameters from modelled data correlate well to the results of AFM imaging. Hence, there is evidence of co-assembly of the fibrous superstructures; given that different monomers may be used to introduce variations into chemical and physical properties, the results demonstrate a self-assembled analogue of a statistical co-polymer that can be used in designing complex functional nanomaterials.

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Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

10.1101/631432 ◽

2019 ◽

Author(s):

Junetha Syed ◽

Saravanan Palani ◽

Scott T. Clarke ◽

Zainab Asad ◽

Andrew R. Bottrill ◽

...

Keyword(s):

Genetic Code ◽

Protein Function ◽

Fluorescent Protein ◽

Trna Synthetase ◽

Glutathione S Transferase ◽

Base Pairs ◽

Site Specific ◽

Green Fluorescent ◽

Natural Amino Acids ◽

Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

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FLUORESCENCE-ACTIVATED CELL SORTING: ISOLATION OF GENETIC VARIANTS FOR THE ANALYSIS OF PROTEIN STRUCTURE AND FUNCTION

Review Articles, including those from an International Conference held in Bielefeld, F. R. of Germany, June 1–2, 1984 ◽

10.1515/9783112393048-005 ◽

1985 ◽

pp. 111-124

Author(s):

Andreas Radbruch

Keyword(s):

Protein Structure ◽

Cell Sorting ◽

Genetic Variants ◽

Structure And Function ◽

Fluorescence Activated Cell Sorting ◽

Protein Structure And Function ◽

And Function

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Two Distantly Homologous DnaG Primases from Thermoanaerobacter tengcongensis Exhibit Distinct Initiation Specificities and Priming Activities

Journal of Bacteriology ◽

10.1128/jb.01511-09 ◽

2010 ◽

Vol 192 (11) ◽

pp. 2670-2681 ◽

Cited By ~ 2

Author(s):

Jie Li ◽

Jingfang Liu ◽

Ligang Zhou ◽

Huadong Pei ◽

Jian Zhou ◽

...

Keyword(s):

Amino Acids ◽

De Novo ◽

Thermophilic Bacterium ◽

Structure And Function ◽

Thermoanaerobacter Tengcongensis ◽

Wide Range ◽

Dna Binding Affinity ◽

Template Specificity ◽

And Function

ABSTRACT Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerase. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG 2, in the thermophilic bacterium Thermoanaerobacter tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibit primase activities in vitro at a wide range of temperatures. Interestingly, the template recognition specificities of these two primases are quite distinctive. When trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on templates containing the trinucleotide 5′-CCC-3′, not on the other 63 possible trinucleotides. When the 5′-CCC-3′ sequence was flanked by additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA primer synthesis on a limited set of tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA primer synthesis more preferably on GC-containing tetranucleotides. In contrast, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates tested. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity in the structure and function of the primases.

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Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms20030492 ◽

2019 ◽

Vol 20 (3) ◽

pp. 492 ◽

Cited By ~ 8

Author(s):

Jiro Adachi ◽

Kazushige Katsura ◽

Eiko Seki ◽

Chie Takemoto ◽

Mikako Shirouzu ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Protein Synthesis ◽

Trna Synthetase ◽

Translation Efficiency ◽

Free System ◽

Free Protein ◽

Cell Free System ◽

Natural Amino Acids ◽

Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

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