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. acknowledges the National Science foundation (DMR-1654405) and Cancer Prevention Research Institute of Texas (CPRIT) (RP170752s) for their support.

1. Preparation
<ol>
	<li>Make Lysogeny broth (LB) agar and pour plates12.</li>
	<li>Transform BL21(DE3) with a pET28 plasmid containing the wtQ&#946; coat protein sequence.
	<ol>
		<li>Thaw E. coli BL21(DE3) competent cells in an ice bath. Place 50 &#956;L of cells in a microcentrifuge tube.</li>
		<li>Add 2 &#956;L of plasmid into one tube and gently flick the tube. Then incubate on ice for 30 min.</li>
		<li>Heat-shock the cells for 45 s in a water bath that is at exa...

<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>

Compared to smaller protein purification, a unique step in purifying bacteriophage Q&#946; is the sucrose gradient centrifugation. After the chloroform/n-butanol extraction step, Q&#946; is further purified using 5-40% sucrose gradients. During centrifugation, particles are separated based on their sizes. Larger particles travel to the higher density region, while smaller particles stay in the lower density region. Q&#946; travels to the lower third of the gradient and remains there while smaller protein impurities are t...

Using nano-sized viral capsids has emerged as an exciting field, which aims to broaden the scope of applications in biomedical research1,2,3. Recombinantly expressed virus-like particles (VLPs) are structurally derived from viruses, but they lack the original viral genetic material making them non-infectious proteinaceous nanoparticles. As the surface features are genetically programed and each capsid is expressed identically to the ones before and after it, it is possible to know the location and number of reactive side chains of the amino acids with atomistic precision. In many cases, both the exterior and interior surfaces possess many kinds of solvent exposed amino acid residues, which can feasibly be functionalized through bioconjugation reactions - reactions that form covalent bonds between a biomolecule and a synthetic molecule4,5.
Bioconjugation reactions help biomolecules of interest have more diverse functionalities in a relatively straightforward fashion. Molecules of interest, such as therapeutic drugs6, fluorescent tags7 and polymers8,9 can be pre-synthesized and characterized before they are attached on the surface of VLPs. A particularly common VLP in biomedical and biomaterials research has been the VLP based upon Bacteriophage Q&#946;, which, as recombinantly expressed, is a 28 nm icosahedral viral capsid10. The most common reaction sites on Q&#946; are lysines by a wide margin, though we have recently communicated the successful conjugation11 of dibromomaleimide compounds to the reduced disulfides that line the pores of Q&#946; via a Haddleton-Baker reaction. The reaction proceeds with good yield and, equally importantly, without losing the thermal stability of the particles. At the same time, this reaction generates conjugation-induced fluorescence, which can be used to track the uptake of these particles into cells. In this work, we demonstrate the conjugation of polyethylene glycol (PEG) onto the surface of Q&#946; through the Haddleton-Baker reaction, which results in a bright yellow fluorophore. These particles can then be tracked as they are taken in by cells. The protocol herein will help researchers generate new fluorescent PEGylated proteinaceous nanoparticles based upon Q&#946;, though its principles are applicable to one of the many other VLPs containing solvent exposed disulfides.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LB Broth (Miller)&#160;</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">1.10285.0500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Tryptone, Poweder</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">T60060-1000.0</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Yeast Extract, Poweder</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">Y20020-1000.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anhydrous magnesium sulfate</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">CI-06808-1KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride (Crystalline/Certified ACS)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Elga PURELAB Flex 3 Water Purification System</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">4474524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Dibasic Anhydrous</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">P288-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S25590B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP2818500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fiberlite F10-4x1000 LEX rotor&#160;</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">096-041053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Sulfate</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">KW-0066-5KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">32614-M6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Butanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">A399-4</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">SW 28 Ti Rotor, Swinging Bucket, Aluminum</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">342204: SW 28 Ti Rotor/ 342217: Bucket Set</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">337922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">PI23200</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">TRIS Hydrochloride</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">T60050-1000.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tetramethylethylenediamine</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">J63734-AC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">C4706-2G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 3-Dibromomaleimide 97%</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">553603-5G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polythylene Glycol</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">41561-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Phosphate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC424375000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylamide/bis-Acrylamide</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">RP-A11310-500.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP179-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FV3000 confocal laser scanning microscope</td>
			<td style="width:71px;">Olympus&#160;</td>
			<td>FV3000&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Labnet Revolver Adjustable Rotator&#160;</td>
			<td style="width:71px;">Thomas Scientific&#160;</td>
			<td>1190P25&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap&#160;</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">010-1459</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3141-0250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Round-bottom tubes; 38 mL; PC</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3117-0380</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty Rim</td>
			<td style="width:71px;">Pyrex</td>
			<td style="width:119px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">UFC801024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M-110P Microfluidizer Materials Processor</td>
			<td style="width:71px;">Microfluidics</td>
			<td style="width:119px;">M-110P</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3117-0380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bottle, with Cap Assembly, Polycarbonate</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">41121703</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cylinder, Graduated - Polypropylene 250 mL</td>
			<td style="width:71px;">PolyLab</td>
			<td style="width:119px;">80005</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">533LS-E Series Steam Sterilizers</td>
			<td style="width:71px;">Getinge</td>
			<td style="width:119px;">533LS-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid</td>
			<td style="width:71px;">LabSource</td>
			<td style="width:119px;">D36-313-CS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 15 mL Conical Centrifuge Tube</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcentifuge Tube: 1.5mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">VWR Os-500 Orbital Shaker</td>
			<td style="width:71px;">VWR Scientifc Products</td>
			<td style="width:119px;">14005-830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetra Handcast systems</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">1658000FC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polypropylene, 250 mL</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">41121703</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Spectrofluorometer NanoDrop</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">3300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Long Needle&#160;</td>
			<td style="width:71px;">Hamilton&#160;</td>
			<td style="width:119px;">7693</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Exel International 5 to 6 cc Syringes Luer Lock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-841-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P200 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P100 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P10 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F144802</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Intel Weighing PM-100 Laboratory Classic High Precision Laboratory Balance</td>
			<td style="width:71px;">Intelligent Weighting Technology</td>
			<td style="width:119px;">IWT_PM100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 50 mL Conical Centrifuge Tube</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4&#8211;15% Mini-PROTEAN TGX Gel, 10 well, 50 &#181;l</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">456-1084</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

The dibromomaleimide derivatives can be synthesized through the condensation reaction between dibromomaleimide anhydride and primary amines15. Alternatively, a mild synthetic method16 using N-methoxycarbonyl activated 3,4-dibromomaleimide was exploited here by reacting with methoxypolyethylene glycol (PEG) to yield DB-PEG (Figure 1). NMR was used to identify the compound structure (Figure 2<...

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

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

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

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

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

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

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 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

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

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