Labeling of Surface-Accessible Cysteine Residues in Engineered Virus-Like Particles

Abstract

Source: Natilla, A.,and Hammond, R. W. Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs. J. Vis. Exp. (2013)

This video demonstrates site-specific fluorescent labeling of mutant virus-like particles (VLPs) produced in Nicotiana benthamiana using a thiol-reactive dye, followed by purification and gel electrophoresis to confirm surface modification.

Protocol

1. Virus Inoculation and VLPs Purification from Nicotiana benthamiana Plants

1. Produce capped T7-RNA transcripts from Potato virus X (PVX)-based vector plasmids carrying Maize rayado fino virus (MRFV) wild-type (wt) and Cysteine-mutated coat protein (CP) genes, using Ambion's T7-mMessage mMachine Kit.
2. For each T7 transcript reaction, inoculate two fully expanded leaves of N. benthamiana with 10 μL reactions and incubate the plants for 10 days in greenhouse, at 60% humidity for 16 h with light (25,000-30,000 lux) at 25 °C and 8 h dark at 20 °C.
3. Harvest N. benthamiana virus-infected leaves 10 days post-inoculation (dpi), weigh the plant tissue (50 grams) and place on ice.
4. Homogenize the plant material in a blender in the presence of 100 mL of a chilled solution of 0.5 M Na-Citrate buffer, pH 5.5 (use 2 mL of buffer per gram of plant tissue).
5. Filter homogenate through 3 layers of cheesecloth and clarify by centrifugation at 27,000 × g for 30 min. Transfer the supernatant into a chilled graduated cylinder, measure the volume, and then transfer to a chilled sterile beaker. Discard the pellet.
6. Process the supernatant by adding 10% polyethylene glycol 8,000 (PEG 8,000), stir for 15 min at 4 °C, and incubate for an additional 45 min at 4 °C to precipitate the virus-like particles (VLPs)-PEG matrix.
7. Centrifuge the sample at 27,000 × g for 20 min at 4 °C and remove the supernatant.
8. Process the pellet by adding 8 mL of 0.05 M Na-Citrate buffer, pH 5.5, containing 2% Triton X-100. Swirl the centrifuge tubes well to release the pellet. Transfer the pellet and buffer to a chilled sterile beaker. Rinse the tubes immediately with 2 mL of the same buffer and add to the beaker. Stir the suspension at 4 °C until the pellets are dissolved (generally 1 h).
9. Clarify by centrifugation for 5 min at 27,000 × g, discard the pellet, and process the supernatant by centrifugation at 140,000 × g for 2 h at 4 °C.
10. Resuspend the pellet in 1 mL 0.05 M Na-Citrate buffer, pH 5.5 and place the suspension on top of a 10-40% sucrose gradient made in 0.05 M Na-Citrate buffer, pH 5.5.
11. Centrifuge the gradient for 3 h at 140,000 × g at 4 °C.
12. Shine a light over the top of the centrifuge tube and collect the light-scattering upper band at 4 cm from the top of the tube, dilute with 0.05 M Na-Citrate buffer, pH 5.5, and centrifuge for 3 h at 140,000 × g at 4 °C.
13. Resuspend the pellet in 0.5 mL of sterile water and store at 4 °C for up to six months.
14. Quantify the VLPs concentration by performing a Bradford protein assay. The yield of VLPs is between 1 and 3 μg/g of plant tissue.

2. Fluorescein-5-maleimide-labeling reactions of VLPs

1. Prepare a 1 mM solution of fluorescein-5-maleimide (427.37 g/mole) in 50 mM sodium phosphate buffer, pH 7.0; 1 mM ethylenediaminetetraacetic acid (EDTA). Mix using a vortex and verify complete dissolution. Protect the tube from light using aluminum foil.
2. Add fluorescein-5-maleimide to VLPs produced in step 1 and mix. Use a 25-fold molar excess of fluorescein-5-maleimide for a molar amount of sulfhydryls. Generally, using 30 μL of VLPs at the concentration of 1 μg/μL and 60 μL of the fluorescein-5-maleimide solution produces acceptable results.
3. Incubate the reaction for 2 hr at room temperature or overnight at 4 °C.
4. Terminate the reaction by adding dithiothreitol (DTT) to a final concentration of 50 mM.
5. Remove unreacted fluorescein using a desalting spin column (Thermo Scientific Inc., Rockford IL) and centrifugation for 2 min at 1,500 × g at room temperature.
6. For Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis, add 10 μL of 2 × SDS-PAGE Laemmli sample buffer to 10 μL of each reaction. Store the labeled protein protected from light at 4 °C for up to one month or in single-use aliquots at -20 °C up to 3 months.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thinwall, Ultra-Clear Tubes	Beckman	344059
mMESSAGE mMACHINE T7 Kit	Life Tecnologies	AM1344M
Fluorescein-5-Maleimide	Thermo Scientific Life Technologies	46130 F150	46130 is out of order substitute with F150
Laemmli Buffer	Bio-Rad	1610737
Tris Glycine SDS (Sodium Dodecyl Sulfate) Running Buffer	Invitrogen	LC2675
Tris Glycine Transfer Buffer	Invitrogen	LC3675
Nitrocellulose Membrane Filter Paper Sandwich	Invitrogen	LC2001
EZ-Link Maleimide-Polyethylene glycol (PEG) 2-Biotin, No-Weigh Format	Thermo Scientific	21901