Name: making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry
Uploaded: 2018-05-27T14:00:00
Description: Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

J.J.G. erkjenner National Science foundation (DMR-1654405) og kreft forebygging Research Institute of Texas (CPRIT) (RP170752s) for deres støtte.

1. forberedelse
<ol><li>Gjør Lysogeny kjøttkraft (LB) agar og hell plater12.</li>
	<li>
Transformere BL21(DE3) med en pET28 plasmider inneholder wtQβ pels protein sekvensen.
	<ol>
<li>Tine E. coli BL21(DE3) kompetent celler i en isbadet. Sted 50 μL av celler i et microcentrifuge rør.</li>
		<li>Legg 2 μL plasmider i en tube og forsiktig sveip røret. Deretter ruge på is 30 min.</li>
		<li>Varme-shock celler for 45 s i et vannbad som er på nøyaktig 42 ° C. Sett...

<ol>
	<li>Pokorski, J., Breitenkamp, K., Liepold, L., Qazi, S., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Virus-Based+Polymer-Protein+Nanoparticles+by+Atom+Transfer+Radical+Polymerization.">Functional Virus-Based Polymer-Protein Nanoparticles by Atom Transfer Radical Polymerization.</a> J. Am. Chem. Soc. 133 (24), 9242-9245 (2011).</li><li>Capehart, S., Coylet, M., Glasgow, J., Francis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Integration+of+Gold+Nanoparticles+and+Organic+Fluorophores+Using+Synthetically+Modified+MS2+Viral+Capsids.">Controlled Integration of Gold Nanoparticles and Organic Fluorophores Using Synthetically Modified MS2 Viral Capsids.</a> J. Am. Chem. Soc. 135 (8), 3011-3016 (2013).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-Directed+Synthesis+of+Porous+and+Protective+Core-Shell+Bionanoparticles.">Template-Directed Synthesis of Porous and Protective Core-Shell Bionanoparticles.</a> Angew. Chem. Int. Ed. 55 (36), 10691-10696 (2016).</li><li>Chen, Z., Li, N., Li, S., Dharmarwardana, M., Schlimme, A., Gassensmith, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Chemistry:+The+Chemical+Functionalization+of+Viral+Architectures+to+Create+New+Technology.">Viral Chemistry: The Chemical Functionalization of Viral Architectures to Create New Technology.</a> WIREs. Nanomed. Nanobiotechnol. 8 (4), 512-534 (2015).</li><li>Chalker, J. M., Bernardes, G. J. L., Lin, Y. A., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+proteins+at+cysteine:+opportunities+in+chemistry+and+biology.">Chemical modification of proteins at cysteine: opportunities in chemistry and biology.</a> Chem. - Asian J. 4 (5), 630-640 (2009).</li><li>Le, D. H., Lee, K. L., Shukla, S., Commandeur, U., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+Virus+X,+a+Filamentous+Plant+Viral+Nanoparticle+for+Doxorubicin+Delivery+in+Cancer+Therapy.">Potato Virus X, a Filamentous Plant Viral Nanoparticle for Doxorubicin Delivery in Cancer Therapy.</a> Nanoscale. 9 (6), 2348-2357 (2017).</li><li>Chen, L., Wu, Y., Yuan, L., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-templated+FRET+Platform+for+the+Rational+Design+of+Ratiometric+Fluorescent+Nanosensors.">Virus-templated FRET Platform for the Rational Design of Ratiometric Fluorescent Nanosensors.</a> Chem. Comm. 51 (50), 10190-10193 (2015).</li><li>Lee, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Structure+and+Conformation+Alter+the+Antigenicity+of+Virus-like+Particle-Polymer+Conjugates.">Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle-Polymer Conjugates.</a> J. Am. Chem. Soc. 139 (9), 3312-3315 (2017).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-Protein+Core-Shell+Nanoparticles+for+Enhanced+Antigen+Immunogenicity.">Polymer-Protein Core-Shell Nanoparticles for Enhanced Antigen Immunogenicity.</a> ACS Macro Lett. 6 (4), 442-446 (2017).</li><li>Brown, S. D., Fielder, J. D., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Hybrid+Bacteriophage+Qbeta+virus-like+particles.">Assembly of Hybrid Bacteriophage Qbeta virus-like particles.</a> Biochemistry. 48 (47), 11155-11157 (2009).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Functionalization+across+Quaternary+Structure+in+a+Virus-+like+Particle.">Fluorescent Functionalization across Quaternary Structure in a Virus- like Particle.</a> Bioconjugate Chem. 28 (9), 2277-2283 (2017).</li><li>. Pouring LB Agar Plates Available from: <a target="_blank" href="https://www.addgene.org/protocols/pouring-lb-agar-plates/">https://www.addgene.org/protocols/pouring-lb-agar-plates/</a> (2016)</li><li>Smith, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Modification,+Bioconjugation,+and+Disulfide+Bridging+Using+Bromomaleimides.">Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides.</a> J. Am. Chem. Soc. 132 (6), 1960-1965 (2010).</li><li>Castaneda, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mild+Synthesis+of+N-functionalised+Bromomaleimides,+Thiomaleimides+and+Bromopyridazinediones.">A Mild Synthesis of N-functionalised Bromomaleimides, Thiomaleimides and Bromopyridazinediones.</a> Tetrahedron Lett. 54 (27), 3493-3495 (2013).</li><li>Fiedler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Mutations+Change+the+Structure+and+Stability+of+a+Virus-+Like+Particle.">Engineered Mutations Change the Structure and Stability of a Virus- Like Particle.</a> Biomacromolecules. 13 (8), 2339-2348 (2012).</li><li>Manzenrieder, F., Luxenhofer, R., Retzlaff, M., Jordan, R., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+Virus-like+Particles+with+Poly(2-oxazoline)s.">Stabilization of Virus-like Particles with Poly(2-oxazoline)s.</a> Angew. Chem. Int. Ed. 50 (11), 2601-2605 (2011).</li><li>Chen, Z., Li, N., Chen, L., Lee, J., Gassensmith, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Functionalized+Bacteriophage+Q&#946;+as+a+Photocaged+Drug+Carrier.">Dual Functionalized Bacteriophage Q&#946; as a Photocaged Drug Carrier.</a> Small. 12 (33), 4563-4571 (2016).</li></ol>

Sammenlignet med mindre protein rensing, er en enestående steg i rensing bacteriophage Qβ sukrose gradient sentrifugering. Etter kloroform/n-butanol utvinning trinn, er Qβ videre renset med 5-40% sukrose graderinger. Under sentrifugering skilles partikler basert på deres størrelser. Større partikler reise til høyere tetthet regionen, mens mindre partikler bo i regionen for lavere tetthet. Qβ reiser til nedre tredjedel av graderingen og fortsatt det mens mindre protein urenheter er fanget på toppen av sentrifuger...

Bruke nano-størrelse viral capsids har dukket opp som et spennende felt, som har som mål å utvide omfanget av programmer i biomedisinsk forskning1,2,3. Recombinantly uttrykt viruslignende partikler (VLPs) er strukturelt avledet fra virus, men de mangler original viral genetiske materialet gjør dem ikke-smittsomme proteinaceous nanopartikler. Idet overflaten funksjonene er genetisk programmerte og hver kapsid uttrykkes identisk til de før og etter det, er det mulig å vite plassering og antall reaktive siden kjeder av aminosyrer med atomistic presisjon. I mange tilfeller har både utvendig og innvendig overflater mange typer løsemidler eksponert aminosyre rester, som kan feasibly være functionalized gjennom bioconjugation reaksjoner - reaksjoner som danner kovalente bindinger mellom en biomolecule og et syntetisk molekyl4,5.
Bioconjugation reaksjoner hjelpe biomolecules rundt har flere ulike funksjoner i en relativt enkel måte. Molekyler steder som terapeutiske narkotika6, fluorescerende tags7 og polymerer8,9 kan pre syntetisert og preget før de knyttes på overflaten av VLPs. En særlig vanlig VLP i biomedisinsk og biologisk materiale forskning har vært VLP basert på Bacteriophage Qβ, som som recombinantly uttrykt, er en 28 nm icosahedral viral kapsid10. De vanligste reaksjon sider på Qβ er lysines av en bred margin om vi nylig har kommunisert på vellykket Bøyning11 dibromomaleimide forbindelser til de reduserte disulfides som porene i Qβ via en Haddleton-Baker reaksjon. Reaksjonen fortsetter med god avkastning, og like viktig, uten å miste den termisk stabiliteten til partikler. Samtidig genererer denne reaksjonen Bøyning-indusert fluorescens, som kan brukes til å spore opptaket av disse partiklene i celler. I dette arbeidet viser vi Bøyning av polyetylenglykol (PEG) på overflaten av Qβ gjennom den Haddleton-Baker reaksjonen, som resulterer i en lys gul fluorophore. Disse partiklene kan deretter spores som de er tatt av celler. Protokollen her vil hjelpe forskerne å generere nye fluorescerende pegylert proteinaceous nanopartikler basert på Qβ, selv om dens prinsipper gjelder for en av de mange andre VLPs inneholder løsemiddel utsatt disulfides.

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			<td style="width:119px;">05-408-129</td>
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making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

Den siste økningen i viruslignende partikler (VLPs) i biomedisinsk og materiale kan tilskrives deres brukervennlighet biosyntesen, diskret størrelse, genetisk programmering og biologisk nedbrytbarhet. Mens de er svært mottakelig for bioconjugation reaksjoner for å legge til syntetiske ligander på overflaten deres, er varierer i bioconjugation metoder på disse vandig født capsids relativt begrenset. For å lette retning av funksjonelle biologisk materiale forskning, må utradisjonelle bioconjugation reaksjoner vurderes. Reaksjonen beskrevet i denne protokollen bruker dibromomaleimides å introdusere ny funksjonalitet i løsemiddelet eksponert disulfide obligasjoner av en VLP basert på Bacteriophage Qβ. Videre er det endelige produktet fluorescerende, som har den ekstra fordelen å generere en sporbar i vitro sonde med en kommersielt tilgjengelig filter.

Dibromomaleimide derivater kan syntetiseres gjennom kondens reaksjonen dibromomaleimide anhydride og primære aminer15. Alternativt ble en mild syntetiske metoden16 bruker N-methoxycarbonyl aktivert 3,4-dibromomaleimide utnyttet her av reagerer med methoxypolyethylene glycol (PEG) til å gi DB-pinne (figur 1). NMR ble brukt til å identifisere sammensatte strukturen (figur 2). Qβ V...

Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Gjør Bøyning-indusert fluorescerende pegylert Virus-lignende partikler av Dibromomaleimide-disulfide kjemi

Her presenterer vi en prosedyre for å fluorescently functionalize disulfides på Qβ VLP med dibromomaleimide. Vi beskriver Qβ uttrykk og rensing, syntese av dibromomaleimide-functionalized molekyler og Bøyning reaksjonen mellom dibromomaleimide og Qβ. Den resulterende gule fluorescerende konjugert partikkelen kan brukes som en fluorescens sonde i cellene.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Avbildning av biologisk vev ved Desorpsjon Elektrospray Ionization massespektrometri

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Intra-lymfeknuterinjeksjon av biologisk nedbrytbare polymerpartikler

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Sirkulasjons mikroRNA Kvantifisering Bruke DNA-bindende Dye kjemi og Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Fastsettelse av Zeta Potensielle via nanopartikkel trans Hastigheter gjennom en fleksibel membran nanopore: Ved hjelp av DNA-modifisert Partikler som et eksempel

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic Plattform med multiplekset Electronic Detection for Spatial Sporing av partikler

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Overlapping av mikro partikler i Nanoplasmonic optisk gitter

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Nettsted rettet immobilisering av bein Morfogenetiske proteinet 2 Solid overflater av Klikk kjemi

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

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