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 exactly 42 &#176;C. Place the tube back in the ice bath immediately after heat-shocking, and incubate for 5 min.</li>
		<li>Add 950 &#956;L of LB media that does not contain any antibiotic.</li>
		<li>Shake the culture at 200 rpm for 60 min at 37 &#176;C.</li>
		<li>Plate 100 &#956;L of the culture on LB agar plates (with Kanamycin) and incubate the plate overnight at 37 &#176;C. Select white colonies when needed.</li>
	</ol>
	</li>
	<li>Make Super Optimal Broth (SOB) media.
	<ol>
		<li>Autoclave two 2 L baffled Erlenmeyer flasks on a superdry cycle.</li>
		<li>In an aseptic environment, weigh out and add 20.0 g of tryptone, 5.0 g of yeast extract, 2.469 g of anhydrous magnesium sulfate, 0.584 g of sodium chloride and 0.186 g of potassium chloride to each flask.</li>
		<li>Bring the volume to 1 L with ultrapure water in each flask and autoclave on the liquid cycle.</li>
		<li>Once the SOB media has reached room temperature after autoclaving, add 1 mL of kanamycin (100 mg/mL) to each liter of media and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Make 0.1 M potassium phosphate buffer (pH 7.00).
	<ol>
		<li>Make 1 M potassium monobasic solution by dissolving 68.045 g of potassium monobasic in 500 mL of ultrapure water.</li>
		<li>Make 1 M potassium dibasic solution by dissolving 87.09 g of potassium dibasic in 500 mL of ultrapure water.</li>
		<li>Add 38.5 mL of potassium monobasic solution and 61.5 mL of potassium dibasic to a 1 L bottle.</li>
		<li>Adjust pH to 7.00 if needed, and bring to a volume of 1 L.</li>
	</ol>
	</li>
	<li>Make 5&#8211;40% sucrose gradients in 0.1 M potassium phosphate buffer (pH 7.00).
	<ol>
		<li>In 50 mL centrifuge tubes, prepare solutions with 5&#8211;40% (increasing in increments of 5%) sucrose dissolved in 0.1 M potassium phosphate buffer (pH 7.00).</li>
		<li>Deposit 3.3 mL of 5% sucrose solution at the bottom of a 38 mL round-bottom polycarbonate tube using a long needle syringe and repeat this for five other tubes.</li>
		<li>Carefully deposit 3.3 mL of 10% sucrose solution at the bottom of the tube, and carefully remove the needle as to not disturb the gradient. Repeat for the other five tubes. 
		&#160;</li>
	</ol>
	</li>
	<li>Continue to deposit 3.3 mL layers of sucrose solutions, increasing from 15% to 40% in each tube, while being cautious to not disturb the gradient.</li>
	<li>When complete, cover the tops of the gradients with foil and store at -80 &#176;C.</li>
</ol>
2. Expression of Q&#946;
<ol>
	<li>Wipe bench area with 1:1 bleach/ethanol.</li>
	<li>Make two 3 mL starter cultures in an aseptic environment by adding single colonies of E. coli BL21(DE3) into 3 mL of SOB media in an aseptic environment.</li>
	<li>Grow on a shaker at 250 rpm in a 37 &#176;C and 0% relative humidity (rH) room overnight.</li>
	<li>Inoculate starter culture in SOB media:
	<ol>
		<li>Take both 3 mL starter cultures off the shaker and, in an aseptic environment, pour each starter culture into one of two 2 L baffled Erlenmeyer flasks with 1 L of fresh SOB media in each.</li>
		<li>Place the inoculated media on a shaker at 250 rpm in a 37 &#176;C and 0% rH room.</li>
	</ol>
	</li>
	<li>Grow the bacteria on a shaker at 250 rpm in a 37 &#176;C and 0% rH room until OD600 reaches 0.9&#8211;1.0.</li>
	<li>Add 1 mL of 1 M isopropyl &#946;-D-1 thiogalactopyranoside (IPTG) using a P1000 pipette to induce protein expression.</li>
	<li>Leave the media on the shaker at 250 rpm in a 37 &#176;C and 0% rH room overnight.&#160;</li>
	<li>Remove media from the shaker the following morning and centrifuge it using 1000 mL bottles at 20,621 &#215; g for 1 h at 4 &#176;C to harvest the cells.</li>
	<li>Discard the supernatant and collect the cell pellet.
	<ol>
		<li>Pour the supernatant into a flask with about 5 mL of bleach to kill bacteria. This is waste.</li>
		<li>Use a spatula to scrape the cell pellet from the bottom of the centrifuge bottle and put the pellet into a 50 mL centrifuge tube.</li>
	</ol>
	</li>
</ol>
3. Purification of Q&#946;
<ol>
	<li>Resuspend the cell pellet with ~20&#8211;30 mL of 0.1 M potassium phosphate buffer (pH 7.00).</li>
	<li>Make sure the resuspension has no chunks, and lyse the cells using a microfluidizer processor according to manufacturer&#8217;s protocol (see Table of Materials). Lyse the cells at least twice to increase the yield of the particles.</li>
	<li>Centrifuge the lysate in 250 mL centrifuge bottles at 20,621 x g for 1 hr at 4 &#176;C.</li>
	<li>Discard the pellet and measure the volume of supernatant in mL. Multiply that value by 0.265 and add that amount of g of ammonium sulfate to the supernatant.</li>
	<li>Stir at 4&#160; &#176;C for at least 1 h on a stir plate at 200 rpm to precipitate out the protein.</li>
	<li>Centrifuge in 250 mL bottles at 20,621 x g for&#160;1 h at 4 &#176;C.</li>
	<li>Discard supernatant and resuspend the pellet with about 10 mL of 0.1 M potassium phosphate buffer (pH 7.00).</li>
	<li>Add equal volumes of 1:1 chloroform/n-butanol to the crude sample and mix by vortexing for a few seconds.</li>
	<li>Centrifuge in 38 mL tubes at 20,621 &#215; g for 30 minutes at 4 &#176;C.</li>
	<li>Recover the top aqueous layer using a Pasteur pipette. Be cautious not to take any of the gel like layer that has formed between the aqueous and organic layer.</li>
	<li>Thaw six 5&#8211;40% pre-made sucrose gradients and load about 2 mL of the extract onto each.</li>
	<li>Ultracentrifuge at 99,582 x g for 16 h at 4 &#176;C with free deceleration.</li>
	<li>Shine a light emitting diode (LED) light under each tube and a blue band should become visible. Recover these particles with a long needle syringe.</li>
	<li>Ultrapellet the particles at 370,541 x g for 2.5 h at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the transparent pellet of purified particles with 0.1 M potassium phosphate buffer (pH 7.00).</li>
</ol>
4. Quantification and Confirmation of the Product
<ol>
	<li>Use Bradford Assay to quantify the product13.</li>
	<li>Run reducing and non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the product14. 
	NOTE: Reducing SDS-PAGE is used to confirm the molecular weight of coat protein; non-reducing SDS-PAGE is to confirm the higher order structure.</li>
</ol>
5. Conjugating DB Compounds on Q&#946;
<ol>
	<li>Reduce disulfides on Q&#946;.
	<ol>
		<li>Dissolve 0.0020 g of tris(2-carboxyethyl)phosphine (TCEP) into 1 mL of ultrapure water to make a 100x stock solution. 
		NOTE: Prepare fresh TCEP before reduction.</li>
		<li>Add 200 &#181;L of Q&#946; (5 mg/mL) into a microcentrifuge tube.</li>
		<li>Follow by adding 20 &#181;L of 100x TCEP stock solution.</li>
		<li>Incubate at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>Re-bridge reduced disulfide using dibromomaleimide polyethylene glycol (DB-PEG).
	<ol>
		<li>Dissolve 0.0017 g of dibromomaleimide-polyethylene glycol (DB-PEG) in 100 &#181;L of DMF.</li>
		<li>Add 680 &#181;L of 10 mM sodium phosphate solution (pH 5.00).</li>
		<li>Add reduced Q&#946; into the solution of DB-PEG and observe the mixing process under a 365 nm UV lamp. The bright yellow fluorescence can be immediately visualized with a 365 nm handheld UV lamp upon mixing.</li>
		<li>Let the reaction proceed overnight at room temperature (RT) on a rotisserie.</li>
	</ol>
	</li>
	<li>Purify the reaction mixture by centrifugal filter (COMW = 10 kDa) three times using 1x PBS at 3,283 x g for 20 min at 4 &#176;C.</li>
	<li>Monitor the conjugation by non-reducing SDS-PAGE and native agarose gel electrophoresis. 
	NOTE: VLPs run as intact particles in native agarose gel, and they are separated based on their charge, size and shape.</li>
</ol>

<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. 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The authors declare that they have no competing financial interests.

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

making-conjugation-induced-fluorescent-pegylated-virus-like-particles

Research

JoVE Journal

Bioengineering

6.8K Views.  University of Texas at Dallas. Here, we present a procedure to fluorescently functionalize the disulfides on Qβ VLP with dibromomaleimide. We describe Qβ expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Qβ. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

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

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Immunology and Infection

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

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

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Visualizing Dengue Virus through Alexa Fluor Labeling

Viral Nanoparticles for In vivo Tumor Imaging

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting