Method Article

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

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DOI:

10.3791/57712

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May 27th, 2018

 ,  ,  , 
1Department of Chemistry & Biochemistry, University of Texas at Dallas, 2Undergraduate Biology, University of Texas at Dallas, 3Undergraduate Healthcare Studies, University of Texas at Dallas, 4Departments of Chemistry & Biochemistry and Biomedical Engineering, University of Texas at Dallas

In This Article

Summary

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.

Abstract

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'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 Qβ. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Introduction

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 Qβ, which, as recombinantly expressed, is a 28 nm icosahedral viral capsid10. The most common reaction sites on Qβ 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 Qβ 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 Qβ 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 Qβ, though its principles are applicable to one of the many other VLPs containing solvent exposed disulfides.

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Protocol

1. Preparation

  1. Make Lysogeny broth (LB) agar and pour plates12.
  2. Transform BL21(DE3) with a pET28 plasmid containing the wtQβ coat protein sequence.
    1. Thaw E. coli BL21(DE3) competent cells in an ice bath. Place 50 μL of cells in a microcentrifuge tube.
    2. Add 2 μL of plasmid into one tube and gently flick the tube. Then incubate on ice for 30 min.
    3. Heat-shock the cells for 45 s in a water bath that is at exactly 42 °C. Place the tube back in the ice bath immediately after heat-shocking, and incubate for 5 min.
    4. Add 950 μL of LB media that does not contain any antibiotic.
    5. Shake the culture at 200 rpm for 60 min at 37 °C.
    6. Plate 100 μL of the culture on LB agar plates (with Kanamycin) and incubate the plate overnight at 37 °C. Select white colonies when needed.
  3. Make Super Optimal Broth (SOB) media.
    1. Autoclave two 2 L baffled Erlenmeyer flasks on a superdry cycle.
    2. 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.
    3. Bring the volume to 1 L with ultrapure water in each flask and autoclave on the liquid cycle.
    4. 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 °C.
  4. Make 0.1 M potassium phosphate buffer (pH 7.00).
    1. Make 1 M potassium monobasic solution by dissolving 68.045 g of potassium monobasic in 500 mL of ultrapure water.
    2. Make 1 M potassium dibasic solution by dissolving 87.09 g of potassium dibasic in 500 mL of ultrapure water.
    3. Add 38.5 mL of potassium monobasic solution and 61.5 mL of potassium dibasic to a 1 L bottle.
    4. Adjust pH to 7.00 if needed, and bring to a volume of 1 L.
  5. Make 5–40% sucrose gradients in 0.1 M potassium phosphate buffer (pH 7.00).
    1. In 50 mL centrifuge tubes, prepare solutions with 5–40% (increasing in increments of 5%) sucrose dissolved in 0.1 M potassium phosphate buffer (pH 7.00).
    2. 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.
    3. 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.
       
  6. 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.
  7. When complete, cover the tops of the gradients with foil and store at -80 °C.

2. Expression of Qβ

  1. Wipe bench area with 1:1 bleach/ethanol.
  2. 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.
  3. Grow on a shaker at 250 rpm in a 37 °C and 0% relative humidity (rH) room overnight.
  4. Inoculate starter culture in SOB media:
    1. 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.
    2. Place the inoculated media on a shaker at 250 rpm in a 37 °C and 0% rH room.
  5. Grow the bacteria on a shaker at 250 rpm in a 37 °C and 0% rH room until OD600 reaches 0.9–1.0.
  6. Add 1 mL of 1 M isopropyl β-D-1 thiogalactopyranoside (IPTG) using a P1000 pipette to induce protein expression.
  7. Leave the media on the shaker at 250 rpm in a 37 °C and 0% rH room overnight. 
  8. Remove media from the shaker the following morning and centrifuge it using 1000 mL bottles at 20,621 × g for 1 h at 4 °C to harvest the cells.
  9. Discard the supernatant and collect the cell pellet.
    1. Pour the supernatant into a flask with about 5 mL of bleach to kill bacteria. This is waste.
    2. 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.

3. Purification of Qβ

  1. Resuspend the cell pellet with ~20–30 mL of 0.1 M potassium phosphate buffer (pH 7.00).
  2. Make sure the resuspension has no chunks, and lyse the cells using a microfluidizer processor according to manufacturer’s protocol (see Table of Materials). Lyse the cells at least twice to increase the yield of the particles.
  3. Centrifuge the lysate in 250 mL centrifuge bottles at 20,621 x g for 1 hr at 4 °C.
  4. 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.
  5. Stir at 4  °C for at least 1 h on a stir plate at 200 rpm to precipitate out the protein.
  6. Centrifuge in 250 mL bottles at 20,621 x g for 1 h at 4 °C.
  7. Discard supernatant and resuspend the pellet with about 10 mL of 0.1 M potassium phosphate buffer (pH 7.00).
  8. Add equal volumes of 1:1 chloroform/n-butanol to the crude sample and mix by vortexing for a few seconds.
  9. Centrifuge in 38 mL tubes at 20,621 × g for 30 minutes at 4 °C.
  10. 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.
  11. Thaw six 5–40% pre-made sucrose gradients and load about 2 mL of the extract onto each.
  12. Ultracentrifuge at 99,582 x g for 16 h at 4 °C with free deceleration.
  13. 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.
  14. Ultrapellet the particles at 370,541 x g for 2.5 h at 4 °C.
  15. Discard the supernatant and resuspend the transparent pellet of purified particles with 0.1 M potassium phosphate buffer (pH 7.00).

4. Quantification and Confirmation of the Product

  1. Use Bradford Assay to quantify the product13.
  2. 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.

5. Conjugating DB Compounds on Qβ

  1. Reduce disulfides on Qβ.
    1. 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.
    2. Add 200 µL of Qβ (5 mg/mL) into a microcentrifuge tube.
    3. Follow by adding 20 µL of 100x TCEP stock solution.
    4. Incubate at room temperature for 1 h.
  2. Re-bridge reduced disulfide using dibromomaleimide polyethylene glycol (DB-PEG).
    1. Dissolve 0.0017 g of dibromomaleimide-polyethylene glycol (DB-PEG) in 100 µL of DMF.
    2. Add 680 µL of 10 mM sodium phosphate solution (pH 5.00).
    3. Add reduced Qβ 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.
    4. Let the reaction proceed overnight at room temperature (RT) on a rotisserie.
  3. 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 °C.
  4. 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.

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Results

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<...

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Discussion

Compared to smaller protein purification, a unique step in purifying bacteriophage Qβ is the sucrose gradient centrifugation. After the chloroform/n-butanol extraction step, Qβ 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. Qβ travels to the lower third of the gradient and remains there while smaller protein impurities are t...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
LB Broth (Miller) EMD Millipore1.10285.0500
Tryptone, PowederResearch Products InternationalT60060-1000.0
Yeast Extract, PowederResearch Products InternationalY20020-1000.0
Anhydrous magnesium sulfateP212121CI-06808-1KG
Sodium Chloride (Crystalline/Certified ACS)Fisher ScientificS271-10
Potassium ChlorideFisher ScientificBP366-500
Elga PURELAB Flex 3 Water Purification SystemFisher Scientific4474524
Potassium Phosphate MonobasicFisher ScientificBP362-1
Potassium Phosphate Dibasic AnhydrousFisher ScientificP288-500
SucroseFisher ScientificS25590B
EthanolFisher ScientificBP2818500
Isopropyl β-D-1-thiogalactopyranoside (IPTG)Sigma AldrichI6758-1G
Fiberlite F10-4x1000 LEX rotor Fisher Scientific096-041053
Ammonium SulfateP212121KW-0066-5KG
ChloroformAlfa Aesar32614-M6
1-ButanolFisher ScientificA399-4
SW 28 Ti Rotor, Swinging Bucket, AluminumBeckman Coulter342204: SW 28 Ti Rotor/ 342217: Bucket Set
Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,Beckman Coulter337922
Coomassie (Bradford) Protein AssayFisher ScientificPI23200
TRIS HydrochlorideResearch Products InternationalT60050-1000.0
TetramethylethylenediamineAlfa AesarJ63734-AC
Tris(2-carboxyethyl)phosphine hydrochlorideSigma AldrichC4706-2G
2 3-Dibromomaleimide 97%Sigma Aldrich553603-5G
Polythylene GlycolAlfa Aesar41561-22
Sodium PhosphateFisher ScientificAC424375000
Acrylamide/bis-AcrylamideP212121RP-A11310-500.0
Sodium dodecyl sulfateSigma AldrichL3771-100G
Ammonium PersulfateFisher ScientificBP179-100
FV3000 confocal laser scanning microscopeOlympus FV3000 
Labnet Revolver Adjustable Rotator Thomas Scientific 1190P25 
1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap Thermo Scientific010-1459
Nalgene Centrifuge Bottles with Caps, Polypropylene CopolymerThermo Scientific3141-0250
Nunc Round-bottom tubes; 38 mL; PCThermo Scientific3117-0380
2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty RimPyrex4980-2L
Amicon Ultra-4 Centrifugal Filter UnitsMillipore SigmaUFC801024
M-110P Microfluidizer Materials ProcessorMicrofluidicsM-110P
Nalgene High-Speed Polycarbonate Round Bottom Centrifuge TubesThermo Scientific3117-0380PK
Bottle, with Cap Assembly, PolycarbonateBeckman Coulter41121703
Cylinder, Graduated - Polypropylene 250 mLPolyLab80005
533LS-E Series Steam SterilizersGetinge533LS-E
TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with LidLabSourceD36-313-CS
Falcon 15 mL Conical Centrifuge TubeFisher Scientific14-959-53A
Microcentifuge Tube: 1.5mLFisher Scientific05-408-129
VWR Os-500 Orbital ShakerVWR Scientifc Products14005-830
Tetra Handcast systemsBio-Rad1658000FC
Polypropylene, 250 mLBeckman Coulter41121703
Spectrofluorometer NanoDropThermo Fisher Scientific3300
Long Needle Hamilton 7693
Exel International 5 to 6 cc Syringes Luer LockFisher Scientific14-841-46
P1000 PipetmanGilsonF123602
P200 PipetmanGilsonF123601
P100 PipetmanGilsonF123615
P20 PipetmanGilsonF123600
P10 PipetmanGilsonF144802
Intel Weighing PM-100 Laboratory Classic High Precision Laboratory BalanceIntelligent Weighting TechnologyIWT_PM100
Falcon 50 mL Conical Centrifuge TubeFisher Scientific14-432-22
4–15% Mini-PROTEAN TGX Gel, 10 well, 50 µlBio-Rad456-1084

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Tags

Bioconjugation MethodDibromomaleimide ChemistryQBeta BacteriophageDisulfide Bond ReductionFluorescent PEGylationProtein PurificationUltracentrifugationSDS PAGE AnalysisTransmission Electron Microscopy

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