Fluorescent Functionalization of Virus-Like Particles via Disulfide Re-bridging

Abstract

Source: Chen, Z., et al. Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry. J. Vis. Exp. (2018)

This video demonstrates the fluorescent functionalization of virus-like particles by re-bridging reduced disulfides with a polyethylene glycol (PEG) linker, producing stable, trackable conjugates suitable for cellular imaging and targeted delivery in biomedical applications.

Protocol

1. Conjugating Dibromomaleimide (DB) Compounds on Bacteriophage 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. Followed 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).
   3. Add reduced Qβ into the solution of DB-PEG and observe the mixing process under a 365 nm UV (ultraviolet) 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 (molecular weight cut-off (MWCO) = 10 kDa) three times using 1x PBS (phosphate-buffered saline) at 3,283 x g for 20 min at 4 °C.
4. Monitor the conjugation by non-reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and native agarose gel electrophoresis.
   NOTE: VLPs (virus-like particles) run as intact particles in native agarose gel, and they are separated based on their charge, size, and shape.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Amicon Ultra-4 centrifugal filter units	Millipore Sigma	UFC801024
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
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