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 Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Nan Shao ◽  
N. Sadananda Singh ◽  
Susan E. Slade ◽  
Alexandra M. E. Jones ◽  
Mohan K. Balasubramanian
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Unnatural Amino Acids ◽  
Building Blocks ◽  
Trna Synthetase ◽  
Cross Linking ◽  
Site Specific ◽  
Specific Manner ◽  
Protein Functions ◽  
A Site

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.

scholarly journals Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi-Hui Wang ◽  
Mu-Lung Jian ◽  
Pei-Jung Chen ◽  
Jo-Chu Tsou ◽  
Le P. Truong ◽  
...  
Keyword(s):  
Click Reaction ◽  
Trna Synthetase ◽  
Delivery Systems ◽  
Her2 Receptor ◽  
Site Specific ◽  
Protein Cage ◽  
Specific Loading ◽  
Wide Range ◽  
Multiple Drugs ◽  
A Site

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.

scholarly journals Simple Preparation of Green Fluorescent Protein Conjugated with ^|^beta;-Cyclodextrin in a Site Specific Manner

2013 ◽  
Vol 29 (8) ◽  
pp. 811-814 ◽  
Author(s):  
Miho SUZUKI ◽  
Yoshihiro ISHIMARU ◽  
Ayumu SAITO ◽  
Koichi NISHIGAKI
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Site Specific ◽  
Beta Cyclodextrin ◽  
Simple Preparation ◽  
Specific Manner ◽  
Green Fluorescent ◽  
A Site

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.

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

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
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.

scholarly journals Osterix Regulates Tooth Root Formation in a Site-specific Manner

2015 ◽  
Vol 94 (3) ◽  
pp. 430-438 ◽  
Author(s):  
T.H. Kim ◽  
C.H. Bae ◽  
J.C. Lee ◽  
J.E. Kim ◽  
X. Yang ◽  
...  
Keyword(s):  
Root Formation ◽  
Tooth Root ◽  
Site Specific ◽  
Specific Manner ◽  
A Site

Degradation of channelopsin-2 in the absence of retinal and degradation resistance in certain mutants

2013 ◽  
Vol 394 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Sybille Ullrich ◽  
Ronnie Gueta ◽  
Georg Nagel
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Aromatic Amino Acids ◽  
Animal Cells ◽  
Functional Protein ◽  
Enhanced Resistance ◽  
Chloride Pump ◽  
Natronomonas Pharaonis

Abstract Channelrhodopsin-2 is a light-gated cation channel from the green alga Chlamydomonas reinhardtii. It is functional in animal cells and therefore widely used for light-activated depolarization, especially in neurons. To achieve a fully functional protein, the chromophore all-trans-retinal is needed. It has not been investigated whether or not the apoprotein is stable without its cofactor until now. Here we show that channelopsin-2 (Chop2, protein without bound retinal) is much more prone to degradation than channelrhodopsin-2 (protein with retinal). Constructs of Chop2 fused to yellow fluorescent protein (Chop2::YFP) in the absence and presence of retinal confirm this observation by exhibiting strongly differing fluorescence. We present mutants of Chop2 with highly increased stability in the absence of retinal. Substitution of threonine 159 with aromatic amino acids causes enhanced resistance to degradation in the absence of retinal, which is confirmed by fluorescence intensity, the increase in photocurrents on the addition of retinal to previously expressed protein, and Western blot analysis. Exchanging threonine 159 with cysteine, however, increases photocurrents due to better binding of retinal, without obvious stabilization against degradation of the retinal-free opsin. We also show that the light-activated hyperpolarizing chloride pump halorhodopsin from Natronomonas pharaonis (NpHR) is not prone to retinal-dependent degradation.

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