scholarly journals Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System

2019 ◽  
Vol 2 (1) ◽  
pp. 16 ◽  
Author(s):  
Wei Gao ◽  
Ning Bu ◽  
Yuan Lu
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Cell Membrane Permeability ◽  
Simple Method ◽  
Free System ◽  
Model Protein ◽  
Green Fluorescent

Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.

Optimization of the posttranslational click modification of proteins

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.

scholarly journals Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study

2016 ◽  
Vol 72 (1) ◽  
pp. 121-130 ◽  
Author(s):  
Andrew B. Dippel ◽  
Gregory M. Olenginski ◽  
Nicole Maurici ◽  
Melanie T. Liskov ◽  
Scott H. Brewer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Structure ◽  
Crystal Structures ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Local Environment ◽  
Loop Region ◽  
Selective Placement ◽  
Green Fluorescent ◽  
Local Protein

The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the β-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root-mean-square deviation (r.m.s.d.) between the crystal structure of wild-type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site-specificity.

scholarly journals Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

2021 ◽  
Author(s):  
Sebasthian Santiago ◽  
Omer Ad ◽  
Bhavana Shah ◽  
Zhongqi Zhang ◽  
Xizi Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Soluble Protein ◽  
Fluorescent Protein ◽  
Specific Activity ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
High Yield ◽  
Unnatural Amino Acid Mutagenesis ◽  
Commercial Strain ◽  
Green Fluorescent

We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical L-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural L-amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Gregory M. Olenginski ◽  
Juliana Piacentini ◽  
Darcy R. Harris ◽  
Nicolette A. Runko ◽  
Brianna M. Papoutsis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Green Fluorescent ◽  
Absorbance Band ◽  
Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

scholarly journals Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids

Molecules ◽  
2017 ◽  
Vol 22 (7) ◽  
pp. 1194 ◽  
Author(s):  
Jordan K. Villa ◽  
Hong-Anh Tran ◽  
Megha Vipani ◽  
Stephanie Gianturco ◽  
Konark Bhasin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Fluorescence Modulation ◽  
Green Fluorescent

Reversible biomechano-responsive surface based on green fluorescent protein genetically modified with unnatural amino acids

2015 ◽  
Vol 51 (1) ◽  
pp. 232-235 ◽  
Author(s):  
Johan Longo ◽  
Chunyan Yao ◽  
César Rios ◽  
Nguyet Trang Thanh Chau ◽  
Fouzia Boulmedais ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Genetically Modified ◽  
Uniaxial Stretching ◽  
Green Fluorescent

Uniaxial stretching of modified GFP “clicked” onto an elastomer leads to a repeatable and reversible decrease of its fluorescence intensity.

scholarly journals Photoreactive Bicyclic Amino Acids as Substrates for MutantEscherichia coliPhenylalanyl-tRNA Synthetases

2004 ◽  
Vol 279 (19) ◽  
pp. 19839-19845 ◽  
Author(s):  
Thomas Bentin ◽  
Ramin Hamzavi ◽  
Johan Salomonsson ◽  
Hervé Roy ◽  
Michael Ibba ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Engineering ◽  
Unnatural Amino Acids ◽  
High Efficiency ◽  
Trna Synthetase ◽  
Double Ring ◽  
Reactive Groups ◽  
Model Protein

Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested forin vitrocharging of tRNAPheand for protein mutagenesis via the phenylalanyl-tRNA synthetase variant αA294G that is able to facilitatein vivoprotein synthesis using a range ofpara-substituted phenylalanine analogues. The results demonstrate that benzofuranylalanine, but not benzotriazolylalanine, is a substrate for phenylalanine tRNA synthetase αA294G, and matrix-assisted laser desorption ionization time-of-flight analysis showed it to be incorporated into a model protein with high efficiency. Thein vivoincorporation into a target protein of a bicyclic phenylalanine analogue, as described here, demonstrates the applicability of phenylalanine tRNA synthetase variants in expanding the scope of protein engineering.

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 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
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