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

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 research^1,2,3. 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 molecule^4,5.

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 polymers^8,9 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.

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.

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<...

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...

Materials

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