Team:TU Darmstadt/Project/VLP Modification
VLP Modification
Introduction
We already showed that Sortase A7M (BBa_K3187028), as well as the Ca2+-dependent mutant Sortase A5M (BBa_K3187016), can modify LPETGG-tagged coat protein (CP‑LPETGG, BBa_K3187000) with a protein of interest presenting the respective tag (polyglycine, polyG, BBa_K3187018). The next challenge was to prove this is also possible with functionally assembled P22‑Virus-like particles (P22-VLPs) made of both CP and scaffold protein (SP, BBa_K3187021). As shown in Fig. 1 we performed different experiments showing that Sortase A7M and Sortase A5M are indeed able to modify Virus-like particles (VLPs).
Figure 1: Scheme of Sortase mediated P22-VLP modification.
Achievements
Cloning of scaffold and coat protein
VLP assemblyin vitro and in vivo
Imaging P22-VLPs
Imaging capsids containing only coat protein
Transmission Electron Microscopy
It is possible to enlarge images up to 150,000 times using a Transmission Electron Microscope (TEM), resulting in a resolution of a few nanometers. An electron beam is focused on the sample through an electromagnetic lens which creates a high-resolution image. A diffraction image is also created through this process, which is especially interesting for crystallographic applications. The beam is diffracted through the coulomb interactions at the core of the atoms. Because of this, a larger atomic mass (mass contrast) or thicker (thick contrast) samples result in a darker image since there is a higher diffraction probability, less electrons are detected. These two factors are considered amplitude contrast. To create a TEM image special sample preparations must be undertaken. The sample must be very thin to avoid too much diffraction, otherwise the resolution becomes worse. If needed, a coating can be applied to protect organic compounds from being destroyed by the intensity of the electron beam.
Ultracentrifugation
An ultracentrifuge is basically a very fast spinning centrifuge that can accelerate speeds up to 1,000,000 xg. In our case, we used the sucrose cushion method to separate the assembled VLPs from the cell lysate.
Preparation of sfGFP for Modification
The coding sequence for sfGFP with the N-terminal TEV site followed by polyG (sfGFP TEV site) was cloned into pACYCT2 via Gibson Assembly. Successful cloning was confirmed by commercial sequencing. The protein was expressed in E. coli BL21 under control of the IPTG inducible T7 promoter and afterwards purified using Strep-tag II in an ÄKTA pure FPLC. After protein expression and purification, a western blot was performed to check for correct protein sizes. Those could be confirmed as sfGFP TEV site is expected at 26.8 kDa and a broad band can be detected slightly lower than 25 kDa. As the proteins are labeled with a marker for the Strep-tag II, we assume that this protein ran further in the SDS gel, probably due to incomplete denaturation.
Figure 2: Western-blot of sfGFP performed with StrepTactin-horseradish peroxidase (GRP) conjugate. Lanes of relevance have been highlighted.
Dynamic Light Scattering (DLS)
DLS is used to determine size and the size distribution as well as the shape of nanoparticles in suspension. Random movement of solved particles – known as Brownian motion – correlates directly to the size and shape of particles. The bigger the particles the lower the Brownian motion. With DLS, you can observe this random movement by another correlation in the following setup: If a sample is irradiated using a laser beam, the light of the beam is scattered. Scattered light corresponds to the detected intensity fluctuation. The faster the particle the stronger the fluctuation. Through Brownian motion scattered light correlates directly with particle size. It is possible to calculate the diffusion coefficient (D). By inserting D into the Stokes-Einstein equation (1) the hydrodynamic diameter (𝐷H) is obtained:
How VLPs were modified
Assembly was achieved by combining purified SP and CP‑LPETGG as described here. Afterwards an equimolar amount of Sortase A5M, as well as sfGFP in tenfold excess to CP‑LPETGG, was added. The fluorescent protein of our choice was sfGFP whose N-terminal methionine was cleaved off with the tobacco etch virus (TEV) protease, leaving an N-terminal polyG-tag as the sortase substrate. The reaction took place for 3 h at 37 °C.
Modification changes Hydrodynamic diameters
When we started to compare sfGFP-modified VLPs with non-modified VLPs using dynamic light scattering (DLS), we expected a difference in hydrodynamic radii because surface modifications should further increase the hydration of the particles as shown in Fig. 3 [1]. As described here, non-modified VLPs showed hydrodynamic diameters of approximately 112.4 nm ± 41.3 nm. In comparison, modified capsids showed an average hydrodynamic diameter of 1446 nm. In general hydrodynamic diameters depend on several properties like polarity and charges as well as size and shape. These properties can be summed up as the electrical properties of the system [2]. In our case, the drastically elevated hydrodynamic diameter of the P22-VLP linked to sfGFP may result from strong hydration since sfGFP is multiply negatively charged [3]. This probably leads to a tremendous charge density all over the surface. Another possible reason could be the formation of sfGFP dimers attached to the VLPs.
Figure 3: Influence of particle charge on hydrodynamic diameter.
In order to demonstrate the integrity of our modified VLPs we used capsids from the same sample for DLS and electron microscopy which confirms the presence of intact VLPs. The size distribution shows that they still pose a monodisperse species, even though their hydrodynamic diameter is increased compared to unmodified VLPs or capsids containing only CP.
Figure 4: Dynamic light scattering analysis. Hydrodynamic diameters of different P22-VLP species.
Imaging modified VLPs
We used ultracentrifugation over a sucrose cushion to separate freshly modified VLPs from monomeric capsid proteins, Sortase A5M, and sfGFP. After ultracentrifugation, a green fluorescent sediment was clearly visible (Fig. 5). This is a strong indication that sortase has attached sfGFP to the VLP exterior, as only assembled VLPs accumulate in the sediment [4]. We then prepared the ultracentrifugation sediment for transmission electron microscopy . Encouragingly, we observed numerous visually intact VLPs.
Figure 5: Sediment containing P22-VLPs modified with sfGFP using SortaseA5M. Sediment was imaged in transmission electron microscope.
References
- ↑ https://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ pharmaceuticals/ viruses-virus-like-particles/ [1]
- ↑ J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2): 155 [2]
- ↑ Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E., DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge shielding prevents aggregation of supercharged GFP variants at high protein concentration. Molecular pharmaceutics, 14(10), 3269-3280. [3]
- ↑ Patterson, Dustin, et al. "Sortase-mediated ligation as a modular approach for the covalent attachment of proteins to the exterior of the bacteriophage P22-Virus-like particle." Bioconjugate chemistry 28.8 (2017): 2114-2124. [4]
