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, ...

The overall goal of this bio-conjugation method is to fluorescently functionalize disulfide containing virus-like particles. This method can help answer key questions in the bio-conjugation reaction for virus-like particles which have a limited number of amino acid residues that can be functionalized. The main advantage of this technique is that by using one reaction new functional groups can be introduced and conjugation-induced fluorophores can be formed at the same time.In addition to QBeta, we believe this reaction can be applied to any virus-like particles that possess disulfide bonds. Prior to starting the procedure for expressing the QBeta bacteriophage, wipe down the bench area with a 1:1 bleach:ethanol solution. In an aseptic environment, make two three-milliliter starter cultures by adding single colonies of E.coli BL21 into SOB media.Grow the cultures in a 37 degrees Celsius and 0%relative humidity room with shaking at 250 rpm overnight. On the following day, remove both three-milliliter starter cultures from the shaker and in an aseptic environment pour each starter culture into a two-liter baffled Erlenmeyer flask with one liter of fresh SOB media. Place the two flasks of inoculated media on a shaker at 250 rpm in the 37 degrees Celsius and 0%relative humidity room.Grow the bacteria until they reach an optical density at 600 nanometers or OD 600 of 0.9 to 1.0. This usually takes about five hours. When the cultures are at the desired OD 600, use a P1000 pipette to add one milliliter of one-molar IPTG to each flask to induce protein expression.Leave the flasks on the shaker in the warm room overnight. On the following morning, remove the flasks from the shaker, transfer the contents of each flask to a one-liter bottle, and centrifuge at 20, 621 times gravity at four degrees Celsius for one hour to harvest the cells. When the centrifugation is done, discard the supernatant by pouring it into a flask with about five milliliters of bleach to kill the bacteria.To collect the cell pellet, use a spatula to scrape the cell pellet from the bottom of the centrifuge bottle and transfer the pellet to a 50-milliliter centrifuge tube. To begin the procedure for QBeta purification, re-suspend each cell pellet with 20 to 30 milliliters of 0.1-molar potassium phosphate buffer, pH 7. Make sure the resuspension has no chunks.Lyse the cells using a microfluidizer processor according to the manufacturer's protocol. Lyse the cells at least twice to increase the yield of the particles. Transfer the lysates to 250-milliliter centrifuge bottles and centrifuge at 20, 621 times gravity at four degrees Celsius for one hour.Measure the volume of the supernatant in milliliters. Multiply that value by 0.265 and then add to the supernatant that amount in grams of ammonium sulfate. Add a stir bar and stir on a stir plate at 200 rpm at four degrees Celsius for at least one hour to precipitate out the protein.Centrifuge in 250-milliliter bottles at 20, 621 times gravity at four degrees Celsius for an hour. Discard the supernatant and re-suspend the pellet with about 40 milliliters of 0.1-molar potassium phosphate buffer, pH 7. Add to the crude sample equal volumes of 1:1 chloroform:n-Butanol and mix by vortexing for a few seconds.Transfer the mixture to a 38-milliliter tube. Centrifuge at 20, 621 times gravity at four degrees Celsius for 30 minutes. Use a pipette to recover the top aqueous layer.Be cautious not to take any of the gel-like layer that has formed between the aqueous and organic layers. Next, thaw six 5 to 40%pre-made sucrose gradients. Load about two milliliters of the extract onto each gradient.Ultra-centrifuge at 99, 582 times gravity at four degrees Celsius for 16 hours with free deceleration. When the ultra-centrifugation is complete, shine a light-emitting diode light under each tube to confirm that a blue band is visible. Use a long needle syringe to recover these particles.Ultra-pellet the particles at 370, 541 times gravity at four degrees Celsius for 2.5 hours. The resulting pellet of purified particles should be transparent. Discard the supernatant and re-suspend the pellet with 0.1-molar potassium phosphate buffer, pH 7.This schematic shows the conjugation that will be demonstrated. 10 equivalents of Tris(2-carboxyethyl)phosphine or TCEP relative to the disulfides in one milligram of QBeta is used to reduce all the disulfides to generate the reduced QBeta capsids at room temperature in one hour. Just before the reduction of disulfides on QBeta, prepare a fresh TCEP solution.Dissolve 0.002 grams of TCEP and one milliliter of ultra-pure water to make a 100X stock solution. Add 200 microliters of five milligrams per milliliter QBeta into a microcentrifuge tube. Then, add 20 microliters of the 100X TCEP stock solution to the microcentrifuge tube.Incubate at room temperature for one hour. In the meantime, prepare the dibromomaleimide polyethylene glycol or DB-PEG solution for the later reaction of re-bridging reduced disulfides. Dissolve 0.0017 grams of DB-PEG in 100 microliters of dimethylformamide.Add 680 microliters of 10-millimolar sodium phosphate solution, pH 5. Next, add the reduced QBeta into the solution of DB-PEG and observe the mixing process under a handheld 365-nanometer UV lamp. Upon mixing, a bright yellow fluorescense should be immediately visible.Let the reaction proceed at room temperature on a rotisserie overnight. On the following morning, purify the reaction mixture by centrifugal filter using 1X PBS at 3, 283 times gravity at four degrees Celsius for 20 minutes. Lastly, monitor the conjugation by non-reducing SDS polyacrylamide gel electrophoresis and native agarose-gel electrophoresis.The conjugation of DB-PEG on QBeta was confirmed by non-reducing SDS polyacrylamide gel electrophoresis under UV and Coomassie blue staining. All the fluorescent bands co-localized with the Coomassie blue staining, representing a successful conjugation. The integrity of QBeta-PEG conjugates was confirmed by native agarose gel electrophoresis and transmission electron microscopy.The top micrograph shows QBeta-maleimide or QBeta-M and the bottom micrograph shows QBeta-PEG. Fluorescence spectroscopy of QBeta-M and QBeta-PEG in 0.1 molar of potassium phosphate buffer showed an excitation maximum around 400 nanometers and an emission maximum around 540 to 550 nanometers. When QBeta-PEG was incubated with mouse macrophage cells in serum-free DMEM medium followed by nucleostaining, co-localization images show that yellow fluorescent particles were taken up by the cells and can be tracked after four hours of incubation.In contrast, the unfunctionalized QBeta virus-like particles show negligible fluorescense. This protocol can help researchers purify QBeta in four days and perform the overnight re-bridging conjugation reaction. While attempting this procedure, it's important to remember that the conjugation reaction works best under acidic conditions, such as pH 5.After watching this video, you should have a good understanding of how to purify QBeta VLP and re-bridge the disulfide bonds with dibromomaleimide compounds to make a fluorescently-labeled VLP. After its development, this technique provided researchers an additional functional handle which can be used to dual-functionalize the exterior surface of bacteriophage QBeta.

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