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. erkänner National Science foundation (DMR-1654405) och Cancer Prevention Research Institute of Texas (CPRIT) (RP170752s) för deras stöd.

1. beredning
<ol><li>Göra Lysogeny buljong (LB) agar och häll tallrikar12.</li>
	<li>
Omvandla BL21(DE3) med en pET28 plasmid som innehåller sekvensen wtQβ coat protein.
	<ol>
<li>Tina E. coli BL21(DE3) behöriga celler i ett isbad. Plats 50 μL av celler i en mikrocentrifug rör.</li>
		<li>Tillsätt 2 μL av Plasmiden i en tub och knacka försiktigt röret. Sedan ruva på is i 30 min.</li>
		<li>Heat-shock cellerna för 45 s i ett vattenbad som är på exakt 42 °...

<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>

Jämfört med mindre protein rening, är en unik steg i rena bacteriophage Qβ den sackaros gradient centrifugeringen. Efter steget kloroform/n-butanol utvinning renas ytterligare Qβ med 5-40% sackaros övertoningar. Under centrifugeringen avskiljs partiklar utifrån deras storlekar. Större partiklar resa till regionen högre densitet, medan mindre partiklar stannar i regionen lägre densitet. Qβ reser till den nedre tredjedelen av lutningen och förblir det medan mindre protein orenheter fångas överst i centrifugr?...

Med hjälp av nano-storlek viral förhoppningsvis har vuxit fram som ett spännande område, som syftar till att utvidga tillämpningsområdet för tillämpningar inom biomedicinsk forskning1,2,3. Recombinantly uttryckt virusliknande viruslika partiklar härrör strukturellt från virus, men de saknar den ursprungliga viral arvsmassa som gör dem icke-infektiös proteinhaltiga nanopartiklar. Som ytstrukturen är genetiskt programmerade och varje kapsid uttrycks identiskt med de före och efter det, är det möjligt att veta platsen och antalet reaktiva sida kedjor av aminosyror med atomistisk precision. I många fall besitter både exteriör och interiör ytorna många sorters lösningsmedel exponerade aminosyra rester, som lämpligen kan vara functionalized genom bioconjugation reaktioner - reaktioner som bildar kovalenta bindningar mellan en biomolecule och en syntetisk molekyl4,5.
Bioconjugation reaktioner hjälpa biomolekyler sevärdheter har mer varierande funktioner på ett relativt enkelt sätt. Molekyler av intresse, såsom terapeutiska läkemedel6, fluorescerande Taggar7 och polymerer8,9 kan vara pre syntetiseras och kännetecknas innan de är fästa på ytan av framställda. Ett särskilt vanligt VLP i biomedicinsk och biomaterial forskning har varit den VLP som baseras på Bacteriophage Qβ, som recombinantly uttryckt, är en 28 nm ikosaedriska virus kapsid10. De vanligaste reaktion webbplatserna på Qβ är lysines med råge, men vi har nyligen kommunicerat framgångsrikt konjugation11 dibromomaleimide föreningar till de reducerade disulfides som kantar porerna i Qβ via en Haddleton-Baker reaktion. Reaktionen fortsätter med bra avkastning och, lika viktigt, utan att förlora den termiska stabiliteten av partiklarna. Samtidigt genererar denna reaktion konjugation-inducerad fluorescens, som kan användas för att spåra spridningen av dessa partiklar in i celler. I detta arbete demonstrera vi konjugationen av polyetylenglykol (PEG) på ytan av Qβ genom Haddleton-Baker reaktion, som resulterar i en ljus gul fluorophore. Dessa partiklar kan sedan spåras när de tas av celler. Protokollet häri hjälper forskare generera nya fluorescerande pegylerat proteinhaltiga nanopartiklar baseras på Qβ, även om dess principer är tillämpliga på en av de många andra framställda som innehåller lösningsmedel exponerade disulfides.

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making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

Den senaste uppgången i virusliknande viruslika partiklar i biomedicinsk och materialforskning kan hänföras till deras användarvänlighet biosyntes, diskreta storlek, genetisk programmering och nedbrytbarhet. Medan de är mycket mottagliga för bioconjugation reaktioner för att lägga till syntetiska ligander på deras yta, är i bioconjugation metoder på dessa aqueous född förhoppningsvis relativt begränsat. För att underlätta ledning av funktionella biomaterial forskning, måste icke-traditionella bioconjugation reaktioner beaktas. Den reaktion som beskrivs i detta protokoll använder dibromomaleimides att införa nya funktioner i lösningsmedlet exponerade disulfide obligationer av en VLP baseras på Bacteriophage Qβ. Slutprodukten är dessutom fluorescerande, som har fördelen av att generera en spårbar in vitro- sonden med hjälp av kommersiellt tillgängliga filter.

Dibromomaleimide derivat kan syntetiseras genom kondensation reaktionen mellan dibromomaleimide bauxit och primära aminer15. Alternativt var en mild syntetisk metod16 använder N-methoxycarbonyl aktiveras 3,4-dibromomaleimide utnyttjas här genom att reagera med methoxypolyethylene glykol (PEG) till avkastning DB-PEG (figur 1). NMR användes för att identifiera den sammansatta strukturen (...

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

Att göra konjugation-inducerad fluorescerande pegylerat virusliknande partiklar av Dibromomaleimide-disulfid kemi

Här presenterar vi ett förfarande för att fluorescently functionalize i disulfides på Qβ VLP med dibromomaleimide. Vi beskriver Qβ uttryck och rening, syntesen av dibromomaleimide-functionalized molekyler och konjugation reaktionen mellan dibromomaleimide och Qβ. Den resulterande gul fluorescerande konjugera partikeln kan användas som en fluorescens sond inuti celler.

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

En mikroflödessystem-baserad Hydrodynamisk fälla för enstaka partiklar

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

Termodynamik för membranprotein Folding Mätt fluorescensspektroskopi

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 biologiska vävnader genom desorption elektrospray masspektrometri

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

Intralymfkörtelinjektion av biologiskt nedbrytbara polymerpartiklar

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

Cirkulerande MicroRNA Kvantifiering Använda DNA-bindande Dye kemi och 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

Fastställande av Zeta Potential via nanopartiklar sloka Hastigheter genom en avstämbar nanopore: Använda DNA-modifierade partiklar som exempel

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

Mikroflödes Plattform med Multiplex elektronisk detektering för rumslig Spårning av partiklar

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

Svällning av mikropartiklar i Nanoplasmonic optiska gallret

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

Webbplats-regisserad immobilisering av benmorfogent Protein 2 till fasta ytor genom att klicka på kemi

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