Editing Qbeta VLP (section)

== [[Category:VLP]]Conjugating DB Compounds on Qβ ==

<br/>1. Reduce disulfides on Qβ.<br/>1. Dissolve 0.0020 g of tris(2-carboxyethyl)phosphine (TCEP) into 1 mL of ultrapure water to make a 100x stock solution.<br/>NOTE: Prepare fresh TCEP before reduction.<br/>2. Add 200 μL of Qβ (5 mg/mL) into a microcentrifuge tube.<br/>3. Follow by adding 20 μL of 100x TCEP stock solution.<br/>4. Incubate at room temperature for 1 h.<br/>2. Re-bridge reduced disulfide using dibromomaleimide polyethylene glycol (DB-PEG).<br/>1. Dissolve 0.0017 g of dibromomaleimide-polyethylene glycol (DB-PEG) in 100 μL of DMF.<br/>2. Add 680 μL of 10 mM sodium phosphate solution (pH 5.00).<br/>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<br/>can be immediately visualized with a 365 nm handheld UV lamp upon mixing.<br/>4. Let the reaction proceed overnight at room temperature (RT) on a rotisserie.<br/>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.<br/>4. Monitor the conjugation by non-reducing SDS-PAGE and native agarose gel electrophoresis.<br/>NOTE: VLPs run as intact particles in native agarose gel, and they are separated based on their charge, size and shape.

&nbsp;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). Qβ VLP is a 28 nm icosahedral proteinaceous nanoparticle, which is composed of 180 identical coat proteins. The coat proteins tend to form noncovalent interlocking dimers through their α-helical domains with the β-sheets from the adjacent coat proteins17. VLPs tend to be selected for their stability at high temperatures, extreme pHs, and in various solvent compositions18. In this case, Qβ VLP is more stable than other RNA phages in the Leviviridac family owing to 180 inter-strand disulfide bonds located at the five- and six- fold axes of symmetry on the capsid19 (Figure 3). These hexameric and pentameric structures are linked by disulfides, which can be visualized by non-reducing SDS-PAGE19. Ten equivalents of TCEP (tris(2-carboxyethyl)phosphine) (0.70 μmol), relative to the disulfides in 1 mg of Qβ (0.070 μmol coat protein, 0.070 μmol disulfides), were used to reduce all the disulfides to generate the reduced Qβ capsids (rQβ) at room temperature in one hour, and non-reducing SDS-PAGE shows that all the higher order structures were reduced to monomeric coat proteins (Figure 4 and Figure 5A). The reaction to rebridge them was done as a one-pot synthesis, as the crude rQβ admixture was added directly to 20 equivalents of a solution of DB-PEG (1.4 μmol) in sodium phosphate (10 mM, pH 5.00, 10% DMF) following the one-hour reduction reaction11. There was observable bright yellow fluorescence under 365 nm UV lamp (Figure 5D) immediately after addition of the DB-PEG. The mixture was then incubated at RT overnight on a rotisserie, followed by purification using centrifugal filter (MWCO = 10 kDa) rinsing with the desired buffer three times to remove excess amounts of small molecules. The Qβ-malemide conjugates were resuspended into 10 mM sodium phosphate solution (pH 5.00) to promote photostability. The conjugation was confirmed by non-reducing SDS-PAGE under UV and coomassie blue staining (Figure 5A). All the bands showed fluorescence (Figure 5A) under UV which colocalized with the coomassie blue staining, representing a successful conjugation. The integrity of Qβ-PEG conjugates were confirmed by native agarose gel electrophoresis and transmission electron microscopy (TEM) (Figure 5B, C). The fluorescence spectroscopy showed the excitation and emission maxima of Qβ-maleimide (Qβ-Μ) and Qβ-PEG to be around 400 nm and 540–550 nm, respectively (Figure 6). This aligns with the commercially available GFP-uv filter set, whose excitation wavelength is 405 nm and emission wavelength is 500–540 nm. The convenient alignment of the photophysical properties of the conjugates with the commercially available filter sets permit using Qβ-PEG as an in vitro probe, which was done and imaged in Figure 7. Qβ-PEG (200 nM) was incubated with Mouse Raw-264.7 cells in serum-free DMEM medium, followed by nucleus staining. Colocalization images in Figure 7 shows that yellow fluorescent particles were uptaken by Raw-264.7 cells and can be tracked after four hours of incubation. The unfunctionalized Qβ VLPs show negligible fluorescence.

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 trapped at the top of the centrifuge tube. The colloidal suspension of Qβ shows strong Tyndall scattering in the sucrose gradient, which can be seen as a blue band when an LED light is shined from underneath. This band is then easy to extract using a long syringe needle. Qβ in sucrose is then pelleted by ultracentrifugation, yielding a clear pellet. The pellet can be resuspended into the desired buffer or further purified by fast protein liquid chromatography (FPLC). The purity of the resulting particle can be confirmed by SDS-PAGE. TCEP is not stable in phosphate buffer, so a 100x TCEP stock solution should be freshly prepared in water right before the reduction. In addition, TCEP neither contains free thiol nor reacts towards other amino acids, so it is not necessary to remove the excess of TCEP prior to performing the conjugation reaction. Dibromomaleimide compounds are dissolved in pH 5.00 sodium phosphate solution, followed by adding the reduced Qβ. At pH 5.00, cysteine (reduced disulfide) is protonated, which promotes the conjugation reaction with dibromomaleimide by preventing reoxidation back to a disulfide. Under 365 nm UV illumination, the reaction mixture emits yellow fluorescence immediately after reduced Qβ was added into DB compounds. Differently from attaching pre-synthesized fluorophores on Qβ, this fluorescence is induced by the conjugation reaction. The fluorophore is formed as soon as the new bonds between sulfur and the maleimide ring are created. Fluorescence spectra shows that the excitation (400 nm) and emission (550 nm) maxima fit the uv-GFP filter set in a confocal microscope (λex = 405 nm, λem = 500−540 nm). Qβ-PEG was then incubated with macrophage Raw 264.7 in serum-free DMEM. After four hours of incubation, Qβ-PEG was uptaken and punctate yellow fluorescent compartments can be visualized inside the cells. One fact that needs to be mentioned is the fluorescence may be transferred for some extent to bovine serum albumin (BSA) in serum or other cysteine-rich substances inside the lysosomes or the late endosomes of cells. Over time, this will weaken the fluorescence inside the cell for cell tracking applications; however, it also provides an opportunity for a new design for drug delivery systems.