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. anerkender National Science foundation (DMR-1654405) og kræft forebyggelse Research Institute of Texas (CPRIT) (RP170752s) for deres støtte.

1. forberedelse
<ol><li>Gøre Lysogeny bouillon (LB) agar og hæld plader12.</li>
	<li>
Omdanne BL21(DE3) med en pET28 plasmid som indeholder wtQβ frakke protein sekvens.
	<ol>
<li>Tø E. coli BL21(DE3) kompetente celler i isbad. Sted 50 μL af celler i et microcentrifuge rør.</li>
		<li>Tilsæt 2 μl plasmid ind i et rør og forsigtigt svirp røret. Derefter inkuberes i isbad i 30 min.</li>
		<li>Heat-shock celler for 45 s i et vandbad, der er på præcis 42 ° C. Pl...

<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. 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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. 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I forhold til mindre protein oprensning, er en unik trin i rensning af bacteriophage Qβ saccharose gradient centrifugering. Efter trinnet chloroform/n-butanol udvinding renses Qβ yderligere ved hjælp af 5-40% saccharose forløb. Under centrifugering adskilles partikler baseret på deres størrelser. Større partikler rejse til regionen højere tæthed, mens mindre partikler bo i regionen Nedre tæthed. Qβ rejser til den nederste tredjedel af gradient og forbliver der, mens mindre protein urenheder er fanget på toppe...

Ved hjælp af nano-størrelse viral capsids fremstod som et spændende felt, som har til formål at udvide anvendelsesområdet for applikationer i biomedicinsk forskning1,2,3. Recombinantly udtrykte viruslignende partikler (VLPs) er strukturelt afledt af virus, men de mangler den oprindelige viral genetiske materiale gør dem ikke-infektiøse proteinholdige nanopartikler. Som de overflade funktioner er genetisk programmerede og hver kapsid udtrykkes identisk til dem før og efter det, er det muligt at kende den placering og antallet af reaktive sidekæder af aminosyrer med atomistisk præcision. I mange tilfælde besidder både de udvendige og indvendige overflader mange slags opløsningsmiddel udsatte aminosyrerester, som realistisk kan være functionalized gennem bioconjugation reaktioner - reaktioner, der danner kovalente bindinger mellem et biomolekyle og syntetisk molekyle4,5.
Bioconjugation reaktioner hjælpe biomolekyler interesse har mere forskelligartede funktioner på en forholdsvis enkel måde. Molekyler af interesse, såsom terapeutiske lægemidler6, fluorescerende tags7 og polymerer8,9 kan pre syntetiseret og karakteriseret før de er fastgjort på overfladen af VLPs. En særlig fælles VLP i biomedicinske og biomaterialer forskning har været VLP baseret på Bacteriophage Qβ, der, som recombinantly udtryk, er en 28 nm ikosaedriske viral kapsid10. De mest almindelige reaktion websteder på Qβ er lysines med en bred margin, selvom vi har for nylig meddelt vellykket konjugation11 af dibromomaleimide forbindelser til de reducerede disulfider, denne linje porerne i Qβ via en Haddleton-Baker reaktion. Reaktionen forløber med godt udbytte, og lige så vigtigt, uden at miste den termiske stabilitet af partikler. På samme tid genererer denne reaktion konjugation-induceret fluorescens, som kan bruges til at spore optagelsen af disse partikler ind i celler. I dette arbejde vise vi konjugation af polyethylenglycol (PEG) på overfladen af Qβ gennem Haddleton-Baker reaktion, hvilket resulterer i en lys gul fluorophore. Disse partikler kan derefter spores, da de er taget af celler. Protokollen heri vil hjælpe forskerne med at generere nye fluorescerende pegyleret proteinholdige nanopartikler baseret på Qβ, selv om dens principper finder anvendelse for en af de mange andre VLPs, som indeholder opløsningsmiddel udsatte disulfider.

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</table>

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

Den seneste stigning i viruslignende partikler (VLPs) i biomedicinsk og materialeforskning kan tilskrives deres lethed af biosyntesen, diskret størrelse, genetisk programmering og bionedbrydelighed. Mens de er meget modtagelig for bioconjugation reaktioner for at tilføje syntetiske ligander på deres overflade, er intervallet i bioconjugation metoder på disse vandig født capsids relativt begrænset. For at lette retning af funktionelle biomaterialer forskning, betragtes som ikke-traditionelle bioconjugation reaktioner. Den reaktion, der er beskrevet i denne protokol bruger dibromomaleimides til at indføre ny funktionalitet i opløsningsmidlet udsatte disulfid obligationer af en VLP baseret på Bacteriophage Qβ. Desuden, det endelige produkt er fluorescerende, som har den fordel at generere en sporbar in vitro- sonden ved hjælp af en kommercielt tilgængelig filtersæt.

Dibromomaleimide derivater kan syntetiseres via kondensationsreaktion mellem dibromomaleimide eddikesyreanhydrid og primære aminer15. Alternativt, en mild syntetiske metode16 ved hjælp af N-methoxycarbonyl aktiveret 3,4-dibromomaleimide blev udnyttet her ved at reagere med methoxypolyethylene glycol (PEG) til udbytte DB-PEG (figur 1). NMR blev brugt til at identificere den sammensatte struktur (<strong class="...

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

Gør konjugation-induceret fluorescerende pegyleret viruslignende partikler af Dibromomaleimide-disulfid kemi

Vi præsenterer her, en procedure for at fluorescently functionalize disulfider på Qβ VLP med dibromomaleimide. Vi beskriver Qβ udtryk og rensning, syntesen af dibromomaleimide-functionalized molekyler og konjugation reaktionen mellem dibromomaleimide og Qβ. Den resulterende gul fluorescerende konjugeret partikel kan bruges som et fluorescens sonde inde i cellerne.

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

Imaging af biologisk væv ved Desorption elektrosprayionisering 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-lymfeknude Injektion af bionedbrydelige 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

Cirkulerende MicroRNA Kvantificering Brug DNA-bindende Dye Kemi 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

Bestemmelse af Zeta Potentielle via Nanopartikel Translokation Hastigheder gennem en Tunable nanopore: Brug af DNA-modificerede Partikler som 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

Mikrofluid Platform med Multiplexed Elektronisk Detection for fysisk sporing af 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

Diffusering af 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

Site-Directed immobilisering af ben morfogenetiske proteiner 2 til faste overflader af klik 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