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

The overall goal of this bio-conjugation method is to fluorescently functionalize disulfide containing virus-like particles. This method can help answer key questions in the bio-conjugation reaction for virus-like particles which have a limited number of amino acid residues that can be functionalized. The main advantage of this technique is that by using one reaction new functional groups can be introduced and conjugation-induced fluorophores can be formed at the same time.

In addition to QBeta, we believe this reaction can be applied to any virus-like particles that possess disulfide bonds. Prior to starting the procedure for expressing the QBeta bacteriophage, wipe down the bench area with a 1:1 bleach:ethanol solution. In an aseptic environment, make two three-milliliter starter cultures by adding single colonies of E.coli BL21 into SOB media.

Grow the cultures in a 37 degrees Celsius and 0%relative humidity room with shaking at 250 rpm overnight. On the following day, remove both three-milliliter starter cultures from the shaker and in an aseptic environment pour each starter culture into a two-liter baffled Erlenmeyer flask with one liter of fresh SOB media. Place the two flasks of inoculated media on a shaker at 250 rpm in the 37 degrees Celsius and 0%relative humidity room.

Grow the bacteria until they reach an optical density at 600 nanometers or OD 600 of 0.9 to 1.0. This usually takes about five hours. When the cultures are at the desired OD 600, use a P1000 pipette to add one milliliter of one-molar IPTG to each flask to induce protein expression.

Leave the flasks on the shaker in the warm room overnight. On the following morning, remove the flasks from the shaker, transfer the contents of each flask to a one-liter bottle, and centrifuge at 20, 621 times gravity at four degrees Celsius for one hour to harvest the cells. When the centrifugation is done, discard the supernatant by pouring it into a flask with about five milliliters of bleach to kill the bacteria.

To collect the cell pellet, use a spatula to scrape the cell pellet from the bottom of the centrifuge bottle and transfer the pellet to a 50-milliliter centrifuge tube. To begin the procedure for QBeta purification, re-suspend each cell pellet with 20 to 30 milliliters of 0.1-molar potassium phosphate buffer, pH 7. Make sure the resuspension has no chunks.

Lyse the cells using a microfluidizer processor according to the manufacturer's protocol. Lyse the cells at least twice to increase the yield of the particles. Transfer the lysates to 250-milliliter centrifuge bottles and centrifuge at 20, 621 times gravity at four degrees Celsius for one hour.

Measure the volume of the supernatant in milliliters. Multiply that value by 0.265 and then add to the supernatant that amount in grams of ammonium sulfate. Add a stir bar and stir on a stir plate at 200 rpm at four degrees Celsius for at least one hour to precipitate out the protein.

Centrifuge in 250-milliliter bottles at 20, 621 times gravity at four degrees Celsius for an hour. Discard the supernatant and re-suspend the pellet with about 40 milliliters of 0.1-molar potassium phosphate buffer, pH 7. Add to the crude sample equal volumes of 1:1 chloroform:n-Butanol and mix by vortexing for a few seconds.

Transfer the mixture to a 38-milliliter tube. Centrifuge at 20, 621 times gravity at four degrees Celsius for 30 minutes. Use a pipette to recover the top aqueous layer.

Be cautious not to take any of the gel-like layer that has formed between the aqueous and organic layers. Next, thaw six 5 to 40%pre-made sucrose gradients. Load about two milliliters of the extract onto each gradient.

Ultra-centrifuge at 99, 582 times gravity at four degrees Celsius for 16 hours with free deceleration. When the ultra-centrifugation is complete, shine a light-emitting diode light under each tube to confirm that a blue band is visible. Use a long needle syringe to recover these particles.

Ultra-pellet the particles at 370, 541 times gravity at four degrees Celsius for 2.5 hours. The resulting pellet of purified particles should be transparent. Discard the supernatant and re-suspend the pellet with 0.1-molar potassium phosphate buffer, pH 7.

This schematic shows the conjugation that will be demonstrated. 10 equivalents of Tris(2-carboxyethyl)phosphine or TCEP relative to the disulfides in one milligram of QBeta is used to reduce all the disulfides to generate the reduced QBeta capsids at room temperature in one hour. Just before the reduction of disulfides on QBeta, prepare a fresh TCEP solution.

Dissolve 0.002 grams of TCEP and one milliliter of ultra-pure water to make a 100X stock solution. Add 200 microliters of five milligrams per milliliter QBeta into a microcentrifuge tube. Then, add 20 microliters of the 100X TCEP stock solution to the microcentrifuge tube.

Incubate at room temperature for one hour. In the meantime, prepare the dibromomaleimide polyethylene glycol or DB-PEG solution for the later reaction of re-bridging reduced disulfides. Dissolve 0.0017 grams of DB-PEG in 100 microliters of dimethylformamide.

Add 680 microliters of 10-millimolar sodium phosphate solution, pH 5. Next, add the reduced QBeta into the solution of DB-PEG and observe the mixing process under a handheld 365-nanometer UV lamp. Upon mixing, a bright yellow fluorescense should be immediately visible.

Let the reaction proceed at room temperature on a rotisserie overnight. On the following morning, purify the reaction mixture by centrifugal filter using 1X PBS at 3, 283 times gravity at four degrees Celsius for 20 minutes. Lastly, monitor the conjugation by non-reducing SDS polyacrylamide gel electrophoresis and native agarose-gel electrophoresis.

The conjugation of DB-PEG on QBeta was confirmed by non-reducing SDS polyacrylamide gel electrophoresis under UV and Coomassie blue staining. All the fluorescent bands co-localized with the Coomassie blue staining, representing a successful conjugation. The integrity of QBeta-PEG conjugates was confirmed by native agarose gel electrophoresis and transmission electron microscopy.

The top micrograph shows QBeta-maleimide or QBeta-M and the bottom micrograph shows QBeta-PEG. Fluorescence spectroscopy of QBeta-M and QBeta-PEG in 0.1 molar of potassium phosphate buffer showed an excitation maximum around 400 nanometers and an emission maximum around 540 to 550 nanometers. When QBeta-PEG was incubated with mouse macrophage cells in serum-free DMEM medium followed by nucleostaining, co-localization images show that yellow fluorescent particles were taken up by the cells and can be tracked after four hours of incubation.

In contrast, the unfunctionalized QBeta virus-like particles show negligible fluorescense. This protocol can help researchers purify QBeta in four days and perform the overnight re-bridging conjugation reaction. While attempting this procedure, it's important to remember that the conjugation reaction works best under acidic conditions, such as pH 5.

After watching this video, you should have a good understanding of how to purify QBeta VLP and re-bridge the disulfide bonds with dibromomaleimide compounds to make a fluorescently-labeled VLP. After its development, this technique provided researchers an additional functional handle which can be used to dual-functionalize the exterior surface of bacteriophage QBeta.