Name: optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells
Uploaded: 2018-04-09T14:30:00
Description: Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

This work has been founded by the Deutsche Forschungsgemeinschaft (DFG) under grants CO822/2-1 (Emmy-Noether program) and CO822/3-1 to I.C.

1. Fluorescence-based Screening of Incorporation Efficiencies (Figure 1)
<ol>
	<li>Maintain HEK293 cells in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM; high glucose, 4 mM glutamine, pyruvate) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 &#181;g/mL streptomycin at 37 &#176;C, 95 % humidity and 5 % CO2.</li>
	<li>Seed the cells the day before transfection.
	<ol>
		<li>Detach the cells for 5 min at 37 &#176;C in 0.05 % Trypsin/PBS supplem...

The protocol describes a simple and reliable assay to assess the efficiency of orthogonal pairs for the incorporation of ncAAs into proteins expressed in mammalian cells. The main advantage of this method in respect to widely used assays based on FACS is that it allows the simultaneous preparation and measurement of larger numbers of samples, and provides data that are easily analyzed using an ordinary software. The availability of a medium-throughput method to analyze orthogonal pairs in mammalian cells is very importan...

The genetic incorporation of chemical probes into proteins is a powerful method to facilitate investigation of structural and dynamic aspects of protein function directly in the native context of the live cell. Nowadays, hundreds of non-canonical amino acids (ncAAs) equipped with the most disparate chemical groups can be site-specifically incorporated into proteins by biosynthesis1,2,3,4. Between them, one finds photo-sensitive ncAAs such as photo-crosslinkers5, photo-caged6,7,8,9 and photo-switchable amino acids10,11, amino acids bearing strained alkenes and alkynes for catalyst-free bioorthogonal chemistry2,12,13,14,15,16,17, amino acids carrying dansyl18, coumarin9,19, and prodan20,21 fluorophores, and amino acids equipped with other biophysical probes as well as with post translational modifications1,2,3,4,22,23,24,25.
The genetic encoding of a ncAA is enabled by a dedicated amino-acyl-tRNA-synthetase (AARS) paired to a cognate suppressor-tRNA, which incorporates the ncAA in response to an amber stop codon during the regular ribosomal synthesis. ncAARS/tRNA pairs are engineered so as to be orthogonal in the host organism, i.e. not cross-talk with the endogenous pairs. The technique is well established both in prokaryotic and eukaryotic hosts and easily applicable to mammalian cells. Pairs for ncAA incorporation in mammalian cells are based on three main orthogonal systems: the tyrosyl system, that combines the TyrRS from E. coli26 with a tyrosyl amber suppressor from B. stearothermophilus27 (EcTyrRS/BstYam pair), the E. coli leucyl system (EcLeuRS/tRNALeuCUA pair)6,18,28 and the archaeal pyrrolysyl system (PylRS/tRNAPyl pair)3, whereby the tRNAPyl is a natural amber suppressor. In general, each ncAA is recognized by a specialized ncAARS. Depending on the structure of the ncAA, the ncAARS is obtained via directed evolution of either TyrRS, LeuRS or PylRS, although some synthetases can accept more than one ncAA.
The orthogonal pair is imported into the cells by simply using a plasmid vector. Most common and efficient plasmids are bicistronic and encode both for the synthetase and the tRNA forming the orthogonal pair29. A second plasmid encoding for the protein of interest bearing an amber codon at the site designated for modification is co-transfected. The ncAA is simply added to the cell growth medium. However, different specialized groups often use different variants of plasmid constructs even for the incorporation of the same ncAA. Constructs differ in the arrangement of the genes in the vector, type of the synthetase, codon usage in the synthetase gene, promoter usage, variant of the tRNA and number of tRNA expression cassettes. Moreover, the incorporation efficiency of different ncAAs can vary drastically due to the different catalytic efficiency of the different synthetases, the quality of the tRNA, and other factors30. Therefore, it is important to have at hand a fast and reliable method to evaluate the efficiency of an orthogonal pair, both to choose the most suitable system for a desired application and to perform some optimization steps that improve overall protein expression yields.
We have established a simple and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs29 (Figure 1). In the assay, cells are co-transfected with the plasmid encoding for the orthogonal pair, together with a bicistronic reporter plasmid encoding both for the green fluorescent protein bearing an amber stop codon at a permissive position (EGFPTAG) and the mCherry gene. Red and green fluorescence of whole-cell lysates are read in separate channels on a plate reader in a 96-well plate. The intensity of the green fluorescence directly correlates with the efficiency of amber suppression, whereas the intensity of red fluorescence gives a direct estimate of the size of the measured sample and the transfection efficiency. With respect to similar assays based on fluorescence assisted cell sorting (FACS) read out31,32, the assay gives an immediate and comprehensive assessment of protein expression in the whole cell population, which is more representative of usual experimental conditions, and offers an easier data acquisition and processing with standard software. Overall, the main advantage of the assay is that a medium to a large number of samples can be analyzed in parallel. Using this assay, we have screened a rationally designed library of suppressor-tRNAs to improve the efficiency of the Pyl orthogonal system30. This work describes the experimental protocol to perform this assay and show examples of its application, including the optimization of the orthogonal pair for the incorporation of the photo-crosslinking ncAA p-azido-L-phenylalanine (Azi) and the comparison of incorporation efficiencies of different amino acids (Figure 2).
Over the last years, ncAA tools have been proven very powerful to investigate structural and functional aspects of G-protein coupled receptors (GPCRs)33,34,35,36,37,38. In humans, GPCRs form a large family of membrane receptors (800 members) and represent main targets for therapeutic drugs. Direct structural characterization of GPCRs is still challenging and complementary biochemical methods are highly needed for their investigation. We have pioneered the use of photo-crosslinking ncAAs to map GPCR surfaces and discover ligand binding pockets34. Using our optimized system for Azi incorporation, we systematically incorporated Azi throughout the whole juxtamembrane domain of a GPCR directly in live mammalian cells. Upon UV irradiation, Azi forms a highly reactive nitrene species that covalently captures neighboring molecules. When the ligand is added to the system, Azi serves as a proximity probe to reveal which positions of the receptor come close to the bound ligand. In this way, the binding mode of the neuropeptide hormone Urocortin I (Ucn1) on the class B GPCR corticotropin-releasing-factor receptor type 1 (CRF1R)33 was first unveiled. Lately, we have disclosed distinct binding patterns of agonists and antagonists on the same receptor38. A similar approach has been applied by others to reveal orthosteric and allosteric binding sites of other peptides and small molecule ligands on other GPCRs39,40,41,42. This manuscript describes the experimental protocol applied in our lab for photo-crosslinking mapping of GPCR surfaces. The method is relatively fast, straightforward and does not require any special equipment, so that it is applicable in standard biochemistry labs. Importantly, the approach provides a valuable tool not only to identify ligand binding sites where 3D structural data are scarce, but also to supplement existing in vitro data with information from fully post-translationally modified receptors in the physiological environment of the live cell.
The recent development of novel ncAAs bearing on the side chain chemical groups suitable for ultrafast catalyst-free bioorthogonal chemistry has opened up the possibility to install last-generation fluorophores for super-resolution imaging into proteins directly on the live cells2,43. Such chemical anchors include strained cyclooctyne in SCOK14, bicyclo[6.1.0]nonyne in BCNK12,17, and trans-cyclooctenes in TCO*K13,15,17 among other ncAAs harboring a norbornene16,17,44 or cyclopropene45,46 moiety. Bulky ncAAs for bioorthogonal chemistry are incorporated by a variant of the PylRS usually denoted as PylRSAF (indicating mutation Y271A and Y349F in M. barkeri PylRS), as well as by other ad hoc evolved ncAARSs17,44. The bioorthogonal anchors react with tetrazine reagents47 via inverse electron-demand Diels-Alder cycloaddition to give high labeling yields within a few minutes43,48. However, application of this powerful approach to label GPCRs has been challenging due to a low overall efficiency of the orthogonal ncAA incorporation system. Using our enhanced Pyl system, we have recently demonstrated high-yield incorporation of such amino acids into GPCRs and ultrafast GPCR labeling on the surface of live mammalian cells30. Labeled receptors were still functional, as they physiologically internalized upon activating the receptor with an agonist. The experimental protocol for the incorporation of bioorthogonal anchors into GPCRs and the following labeling steps are described here. Equipping GPCRs with small bright fluorophores is the first fundamental step toward the study of GPCR structural dynamics in the live cell via advanced microscopy techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/Bisacrylamide 30% (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.2</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Azidophenylalanine (Azi)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenolblue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)</td>
			<td>Carl Roth</td>
			<td>8076.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbobenzyloxy-L-lysine (Lys(Z))</td>
			<td>NovaBiochem</td>
			<td>8540430100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctyne-L-lysine (SCOK)</td>
			<td>Sichem</td>
			<td>SC-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth</td>
			<td>A994.2</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Carl Roth</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>enhanced chemiluminescence reagent (ECL)&#160;</td>
			<td>home-made</td>
			<td></td>
			<td>10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l&#160; p-coumaric acid in DMSO ; 30&#160;% H2O2 (1,000 : 100 : 0.3) [Quelle Laborjounal]</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.1</td>
			<td></td>
		</tr>
		<tr>
			<td>endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)</td>
			<td>Sichem</td>
			<td>SC-8014</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroBrite DMEM</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>7533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth</td>
			<td>9105.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Applichem</td>
			<td>A2185,0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>0082.3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Lactate</td>
			<td>Sigma-Aldrich</td>
			<td>71718-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Gr&#252;ssing</td>
			<td>121551000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Coumaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C9008-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Corning</td>
			<td>354210</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11548876 (15140-122)</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Carl Roth</td>
			<td>6367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F</td>
			<td>NEB</td>
			<td>P0704L</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk Powder&#160;</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Carl Roth</td>
			<td>CN30.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazine-Cy3</td>
			<td>Jena Bioscience</td>
			<td>CLK-014-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-Cyclooctene-L-lysine (TCO*K)</td>
			<td>Sichem</td>
			<td>SC-8008</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5%</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Carl Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wasserstoffperoxid (30%)</td>
			<td>Merck</td>
			<td>1.07210.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-305</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-635</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker Bio-Link 365 nm</td>
			<td>Bio-Budget Technologies GmbH</td>
			<td>40-BLX-E365</td>
			<td>5 x 8 Watt tubes</td>
		</tr>
		<tr>
			<td>Plate Reader BMG LABTECH FLUOstar Omega&#160;</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid E2AziRS</td>
			<td>The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)</td>
			<td></td>
			<td>Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter</td>
		</tr>
		<tr>
			<td>POI-TAG mutant plasmids</td>
			<td></td>
			<td></td>
			<td>Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position&#160;</td>
		</tr>
		<tr>
			<td>CRF1R-95TAG-EGFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>HA-PTH1R-79TAG-CFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Arrestin3-FLAG</td>
			<td>Synthesized by Genart (Life Technologies)</td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FLAG-HRP M2 antibody conjugate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A8592&#160;</td>
			<td>monoclonal, produced in mouse clone M2</td>
		</tr>
		<tr>
			<td>Goat-anti-rabbit-HRP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit-anti-CRF antibody</td>
			<td>home-made</td>
			<td>PBL #rC69</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
		<tr>
			<td>Rabbit-anti-Ucn1 antibody</td>
			<td>home-made</td>
			<td>PBL #5779</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
	</tbody>
</table>

optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

The outline of the fluorescence assay is depicted in Figure 1. The assay is employed in three applications. In first place, a number of tRNA variants for incorporation of Lys(Boc) by the Pyl orthogonal pair are screened. Lys(Boc) is an amino acid sterically similar to Pyl. As Pyl is not commercially available, Lys(Boc) is commonly used as a standard substrate for the PylRS. The screened tRNAs are based on the tRNAPyl. Each tRNA variant bears mutati...

Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

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Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...