This research was supported by the National Institutes of Health NIGMS (R01-GM094541) and Boston College.

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All authors declare no competing financial interests in this manuscript.

Im ersten Teil dieses Protokolls, beschrieben wir eine modifizierte Synthese von 1,2-azaborines basierend auf zuvor beschriebenen Verfahren 12, 13. Triallylborane 22 wurde als Ersatz für die Strecken verwendet allyltriphenyl Zinn oder Kalium allyltrifluoroborate Verwendung zur Herstellung von N -allyl- N -TBS- B allyl Chlorid - Addukt (1). Diese Methode ermöglicht eine atomökonomische und umw...

Bor-Stickstoff - haltige Heterocyclen (dh 1,2-azaborines) wurden kürzlich große Aufmerksamkeit als Isostere von Arene gezogen. Diese Isosterie kann zur Diversifizierung der bestehenden Strukturmotive führen die chemischen Raum 2, 3, 4 zu erweitern. Azaborines haben potentielle Brauchbarkeit für die Anwendung in der biomedizinischen Forschung 5, 6, 7, 8, vor allem im Bereich der medizinischen Chemie in denen Chemiker strukturell Synthese von Bibliotheken durchzuführen und funktionell relevante Moleküle. Bezeichnenderweise jedoch während zahlreiche gut entwickelte Synthesewege zur Verfügung Aren enthaltenden Molekülen gibt es nur eine begrenzte Anzahl von Verfahren für die Synthese von azaborines 9 gemeldet wurden, 10, </sbis&gt; 11, 12, 13. Dies ist vor allem auf eine begrenzte Anzahl von Optionen für die Bor-Quelle und die luft- und feuchtigkeitsempfindlichen Natur des Moleküls in der frühen Phase der Synthesesequenz. Im ersten Teil dieses Artikels, werden wir eine Multi-Gramm - Maßstab Synthese von N -TBS- B -Cl-1,2-Azaborins (3) unter Verwendung von Standardluftfreien Techniken beschreiben. Diese Verbindung dient als vielseitiges Zwischenprodukt , das weiter funktionalisiert werden kann 14 bis strukturell komplexeren Molekülen, 15. Ausgehend von 3, die Synthese und Reinigung von N -H- B -ethyl-1,2-Azaborins (5) zur Verwendung in Protein - Bindungsstudien beschrieben. Aufgrund der Volatilität von 5, erfordert die effiziente Isolierung eine präzise Steuerung der Reaktionstemperatur, Zeit und distillation Bedingungen. Im zweiten Teil, Protokolle für die Proteinexpression und Isolierung von T4 - Lysozym - Mutanten (L99A und L99A / M102Q) 17, 18, 19, 20 wird durch die Proteinkristallisation und Herstellung von Protein-Ligand - Kristallkomplexe dargestellt werden, gefolgt. T4 - Lysozym - Mutanten L99A und L99A / M102Q wurden als biologische Modellsysteme ausgewählt , um die Wasserstoffbindungsfähigkeit von NH enthält Azaborins Moleküle 17 zu untersuchen. Mit einem Standard - Molekularbiologie - Protokoll wird das Protein in Escherichia coli RR1 - Stamm exprimiert und induziert mit Isopropyl-β-D-1-thiogalactopyranosid (IPTG). Proteinreinigung wird Ionenaustausch-Säulenchromatographie durchgeführt werden. Protein Kristallisation wird mit hochkonzentrierten gereinigte Proteinlösung (&gt; 95% Reinheit durch Gelelektrophorese) durchgeführt unter Verwendung des HängeDrop Dampfdiffusionsverfahren. Wegen der Empfindlichkeit dieser Studie Liganden an Sauerstoff, die Protein-Ligand-Komplexe werden unter luftfreien Bedingungen hergestellt.

<table><tbody><tr><td>Tetrahydrofuran (THF), inhibitor-free, for HPLC, &amp;ge;99.9%</td><td>Sigma Aldrich</td><td>34865</td><td></td></tr><tr><td>Diethyl ether (Et&lt;sub&gt;2&lt;/sub&gt;O), for HPLC, &amp;ge;99.9%, inhibitor-free</td><td>Sigma Aldrich</td><td>309966</td><td></td></tr><tr><td>Methylene chloride&amp;nbsp; (CH&lt;sub&gt;2&lt;/sub&gt;Cl&lt;sub&gt;2&lt;/sub&gt;), (Stabilized/Certified ACS)</td><td>Fisher</td><td>D37-20</td><td></td></tr><tr><td>Toluene</td><td>Fisher</td><td>T290-4</td><td></td></tr><tr><td>Pentane, HPLC</td><td>Fisher</td><td>P399-4</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A21-4</td><td></td></tr><tr><td>Calcium hydride (CaH&lt;sub&gt;2&lt;/sub&gt;), reagent grade, 95%</td><td>Sigma Aldrich</td><td>208027</td><td>Pyrophoric</td></tr><tr><td>Palladium on activated carbon (Pd/C), 10 wt% Pd</td><td>Strem</td><td>46-1900</td><td></td></tr><tr><td>1.0 M Boron trichloride solution in hexane</td><td>Sigma Aldrich</td><td>211249</td><td>Highly toxic/Pyrophoric</td></tr><tr><td>Triethylamine, &amp;ge;99.5%</td><td>Sigma Aldrich</td><td>471283</td><td></td></tr><tr><td>Grubbs 1&lt;sup&gt;st&lt;/sup&gt; generation catalyst&amp;nbsp;</td><td>materia</td><td>C823</td><td></td></tr><tr><td>Acetamide</td><td>Sigma Aldrich</td><td>A0500</td><td></td></tr><tr><td>n-Butanol, anhydrous, 99.8%</td><td>Sigma Aldrich</td><td>281549</td><td></td></tr><tr><td>Ethyllithium solution, 0.5&amp;nbsp;M in benzene/cyclohexane</td><td>Sigma Aldrich</td><td>561452</td><td>Highly toxic/ Pyrophoric</td></tr><tr><td>HCl solution, 2.0 M in Et&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>455180</td><td></td></tr><tr><td>2-Methylbutane, anhydrous, &amp;ge;99%</td><td>Sigma Aldrich</td><td>277258</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;, (Migula) Castellani and Chalmers (ATCC&amp;reg; 31343&amp;trade;)</td><td>ATCC</td><td>31343</td><td></td></tr><tr><td>T4 lysozyme WT* (L99A)</td><td>Addgene</td><td>18476</td><td></td></tr><tr><td>T4 lysozyme mutant (S38D L99A M102Q N144D)</td><td>Addgene</td><td>18477</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A0166</td><td></td></tr><tr><td>isopropyl-&amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Invitrogen</td><td>AM9464</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>Fisher</td><td>BP329</td><td></td></tr><tr><td>Sodium Phosphate dibasic anhydrous</td><td>Fisher</td><td>BP332</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>S642212&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher</td><td>BP118</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M4880</td><td>Corrosive</td></tr><tr><td>Thermo scientific pierce DNaseI</td><td>Fisher</td><td>PI-90083</td><td></td></tr><tr><td>GE Healthcare&amp;nbsp;Sepharose Fast Flow Cation Exchange Media</td><td>Fisher</td><td>45-002-931</td><td></td></tr><tr><td>Tris-base</td><td>Fisher</td><td>BP152-500&amp;nbsp;</td><td></td></tr><tr><td>Sodium azide</td><td>TCI</td><td>S0489</td><td>Highly toxic</td></tr><tr><td>2-Mercaptoethanol</td><td>Fisher</td><td>ICN806443&amp;nbsp;</td><td></td></tr><tr><td>Sartorius&amp;nbsp;Vivaspin 20 Centrifugal Concentrators</td><td>Fisher</td><td>14-558-501</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>2-Hydroxyethyl disulfide</td><td>Sigma Aldrich</td><td>380474</td><td></td></tr><tr><td>N-paratone&amp;nbsp;</td><td>Hampton Research</td><td>HR2-643</td><td></td></tr><tr><td>4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO&amp;nbsp;</td><td>Fisher</td><td>08-667E</td><td></td></tr><tr><td>&amp;nbsp;CryoLoop</td><td>Hampton Research</td><td></td><td>cryogenic tubing shaped into a loop</td></tr><tr><td>CryoTong</td><td>Thermo Fisher</td><td></td><td>cryogenic tong</td></tr><tr><td>Coot</td><td></td><td></td><td>Electron density images are generated from the software</td></tr></tbody></table>

synthesis of 1,2-azaborines and the preparation of their protein complexes with t4 lysozyme mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

Die schematische Syntheseweg für 1,2-azaborines ist in Abbildung 1 dargestellt. Dieses Protokoll gilt für die Synthese von fünf verschiedenen Bor-Stickstoff-enthaltende Moleküle. 2 stellt 11 B - NMR - Spektren im Verlauf von Schritt 1.3 gemessen , um die Bildung des gewünschten Produkts (3) zu überwachen. Proteinreinigung wurde unter Verwendung von Niederdruck - Chromatographie - System durchgeführt , und ein repräsent...

Watch this Scientific Journal Video about Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants at JoVE.com

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

Synthese von 1,2-Azaborines und die Vorbereitung ihrer Proteinkomplexe mit T4-Lysozym Mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

synthesis-12-azaborines-preparation-their-protein-complexes-with-t4

Research

JoVE Journal

Biochemistry

7.5K Aufrufe.  Boston College. A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

Serie: Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Synthese von Protein Biokonjugate<em&gt; über</em&gt; Cystein-maleimid Chemie

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Forschung

Chemie

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

Analyse dynamischer Proteinkomplexe montiert auf und befreit Biolayer Interferometrie Biosensor mit Massenspektrometrie und Elektronenmikroskopie

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Biochemie

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Chemoselektive Modifizierung von Oberflächen durch Viral Bioorthogonale Click-Chemie

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunologie und Infektion

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Vorbereitung der stabile bicyclische Aziridinium Ionen und ihre Ring-Öffnung für die Synthese von Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1-Vermittelte enzymatische Synthese und Immobilisierung von polymerisiertem Protein für die Einzelmolekül-Kraftspektroskopie

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

Herstellung von enantiopuren nicht-aktivierten Aziridinen und Synthese von Biemamid B, D und Epiallo-Isomuscarin

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Engineering-"Golden" Fluoreszenz durch Selektionsdruck Einbeziehung der nicht-kanonischen Aminosäuren und Protein-Analyse durch Massenspektrometrie und Fluoreszenz

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Biotechnik

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Sequenzspezifische Markierung von Nukleinsäuren und Proteinen mit Methyltransferasen und Cofaktor-Analoga

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Biologie

<meta charset="utf8"></head><body>Optimiertes Protokoll für die Herstellung von Leishmania Translational Extract (LTE)

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Proteinproduktion mit dem eukaryotischen zellfreien Proteinexpressionssystem LTE

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

Produktion und Optimierung von LTE, einem von Leishmania tarentolae abgeleiteten zellfreien Proteinexpressionssystem für die rekombinante Proteinproduktion

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

Synthese von 1,2-Azaborines und die Vorbereitung ihrer Proteinkomplexe mit T4-Lysozym Mutants

Zusammenfassung

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Chemoselektive Modifizierung von Oberflächen durch Viral Bioorthogonale Click-Chemie

Vorbereitung der stabile bicyclische Aziridinium Ionen und ihre Ring-Öffnung für die Synthese von Azaheterocycles

OaAEP1-Vermittelte enzymatische Synthese und Immobilisierung von polymerisiertem Protein für die Einzelmolekül-Kraftspektroskopie

Herstellung von enantiopuren nicht-aktivierten Aziridinen und Synthese von Biemamid B, D und Epiallo-Isomuscarin

Engineering-"Golden" Fluoreszenz durch Selektionsdruck Einbeziehung der nicht-kanonischen Aminosäuren und Protein-Analyse durch Massenspektrometrie und Fluoreszenz

Sequenzspezifische Markierung von Nukleinsäuren und Proteinen mit Methyltransferasen und Cofaktor-Analoga

Produktion und Optimierung von LTE, einem von Leishmania tarentolae abgeleiteten zellfreien Proteinexpressionssystem für die rekombinante Proteinproduktion

Produktion und Optimierung von LTE, einem von Leishmania tarentolae abgeleiteten zellfreien Proteinexpressionssystem für die rekombinante Proteinproduktion