Team:TU Darmstadt/Project/P22 VLP
VLP Assembly
Introduction
For Virus-like particle (VLP) assembly both capsid proteins of the bacteriophage P22, the
coat protein (CP) (BBa_K3187000 and BBa_K3187001)
and the scaffold protein (SP) (BBa_K3187002 and BBa_K3187003), are needed
[1]
[2]
.
CP and SP then independently assemble through affinity of the proteins to each other.
For our modular platform we wanted to modify the coat proteins with a version of the
Sortase A.
In order to do so, the gene of the CP was extended by a sequence to display the sortase recognition tag, LPETGG,
at the protein's C-terminus.
By fusing the N-terminus of SP with a cargo-protein, we can load the inside of the assembled capsids
with said cargo.
[3]
We used superfolder Green Fluorescent Protein (sfGFP) as a cargo-protein for proof of
concept experiments.
For our project we build our own VLPs, both in vivo and in vitro. We were able to build,
purify, and modify these P22-VLPs.
Achievements
VLP assembly in vitro and in vivo
Imaging of P22-VLPs
Imaging of capsids with only coat protein
Cloning of scaffold protein and coat protein
Confirming the difference in size of VLPs and CP-only-capsids
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 atom. 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, which muddles the resolution. If needed a coating can be applied to protect organic compounds from being destroyed by the intensity of the electron beam.
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 laser 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 to the 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:
Ultracentrifugation
An ultracentrifuge is basically a very fast spinning centrifuge that can accelerate speeds up to 1,000,000 g. In our case we used the Sucrose Cushion method to separate the assembled VLPs from cell lysate or monomeric protien.
VLP forming Proteins
Results Coat protein
After cloning the gene for CP-LPETGG in pET24 via restriction and ligation, the gene was expressed in E. coli BL21 (DE3) using the Isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible T7 promoter and resulting protein purified using its Strep-tag II in a ÄKTA pure fast protein liquid chromatography (FPLC). Similarly, the gene for CP without LPETGG-tag was constructed and afterwards expressed and purified. Correct cloning of the part was verified through sequence analysis. After protein expression and purification, western blot was performed to check for correct protein sizes.
Results Scaffold protein
We cloned a fusion protein of SP and sfGFP into the vector pACYCT2 via gibson assembly. For another experiment, we needed scaffold protein without sfGFP. We deleted the sfGFP part from the plasmid with PCR using overhang primers and restriction/ligation, keeping the pACYCT2 vector. We transformed and expressed scaffold protein with and without sfGFP in E. coli BL21 (DE3) using the IPTG inducible T7 promoter and purified the protein with a Strap-tag II column using ÄKTA pure FPLC. Correct cloning of the part was verified through sequence analysis. After protein expression and purification, western blot was performed to check for correct protein sizes (Fig. 1).
Figure 1: Western blot of expressed proteins. Lanes that are not of interest have been faded out.
CP-LPETGG and CP (without tag) are expected to have a molecular weight of
47 kDa. Respective bands
can be seen on the western blot.
The fusion protein of SP with sfGFP has a molecular weight of 46.1 kDa. Bands
of about this size
can be found on the western blot.
Two additional bands can be observed. One at est. 25 kDa and one between 25 and
37 kDa. The
lower band may be sfGFP and
upper band scaffold protein. We came to this conclusion by comparing the lane of
sfGFP-SP with lanes of
only SP and with sfGFP with TEV cleavage site.
Those two bands are probably produced by the denaturing of the sfGFP-SP fusion
protein. During
denaturation for SDS-PAGE sample preparation,
the fusion protein can break in two parts, sometimes it breaks in front and
sometimes after the
Strep-tag II.
This is indicated by the fact that both bands are stained by an anti-Strep-tag II
western blot.
The band of Strep-tag and SP can be observed at a size of est. 30 kDa. This is
larger than the
expected, theoretical size of SP at about 18 kDa.
Because the plasmid used for expression was verified by sequencing before, and the
fusion protein has
the right size when it is not broken from sample preparation,
we suspect that the protein is the right one and it just behaves unexpected in this
SDS-PAGE.
Assembling P22-VLPs in vivo
Producing P22-VLPs in E. coli
For in vivo capsid production E. coli was cotransformed with two plasmids containing CP and SP respectively. For fluorescent capsids, sfGFP-SP fusion-protein on pACYCT2 and CP-LPETGG on pET24 were used. Produced proteins would then intracellularly self-assemble into functional VLPs.
Figure 2: P22-VLPs assembling inside E. coli.
Concentrating VLPs by Ultracentrifugation
Figure 3: Cell broth after ultracentrifugation. Supernatant containing sfGFP-SP and CP while VLPs collected in the sediment.
For extracting the VLPs directly from cell broth we first lysed the cells by sonication and got rid of debris by two centrifugation steps at 12,000 x g. Afterwards ultracentrifugation on a sucrose cushion (35% w/v) at 150,000 x g was used as a first concentration step. The resulting sediment contained fluorescent material (see Fig. 3) which we suspected to contain a concentrated fraction of VLPs.
Further chromatographic Purification
Ultracentrifugation sediment most likely still contains monomeric proteins and small amounts of cell debris that can be harmful in some applications due to high endotoxin levels. As we revealed in our collaboration work at the Paul-Ehrlich-Institut , endotoxin levels in the ultracentrifugation sediment are indeed extremely high. For getting rid of these contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex-100 column). After SEC the eluted sample with the highest suspected VLP concentration (based on UV absorption) was imaged with transmission electron microscopy (TEM). Numerous capsids in the correct size of 60 nm were clearly visible as can bee seen in Fig. 4. This lead us to believe that ultracentrifugation, as well as SEC treatment, do not interfere with capsid integrity while separating VLPs from other contaminants. Chromatography dilutes the sample significantly which is not optimal for analytic purposes. This is why a second ultracentrifugation treatment would be required for re-concentration of purified capsids as Patterson et al. 2012 suggested [3] .
Figure 4: Intact P22-VLPs after size exclusion chromatography.
Endotoxins in in vivo Production
Endotoxin levels of P22-VLP produced in vivo and in vitro were measured with Limulus amebocyte lysate (LAL) test after ultracentrifugation purification. In vitro produced P22-VLPs showed low Endotoxin levels while VLPs that have been produced in vivo had higher Endotoxin levels (see Fig. 5). This supports the importance of further purification after ultracentrifugation.
Figure 5: Endotoxin levels in endotoxin units/ml (EU/ml) and pg/ml in set volume of ultracentrifugation sediment.
In vitro Assembly of Virus-like Particles
Ultracentrifugation separates VLPs from monomeric protein
In vitro assembly of VLPs was performed by combining sfGFP-SP fusion protein and CP in a molar ratio of 1:2.8. Afterwards possible remaining monomeric proteins were separated from assembled capsids by ultracentrifugation at 150,000 x g on a sucrose cushion (35% w/v) according to (Patterson et al. 2012) [3] . After completion of the ultracentrifugation treatment, fluorescent sediment was clearly visible in the centrifuge tube which we suspected to mainly contain VLPs. Transmission electron microscopy (TEM) was used to image capsids taken from the sediment. For increased contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually intact VLPs all over the sample measuring a diameter that seems to be in the range of 60 nm (see Fig. 6).
Figure 6: Sample after ultracentrifugation. Fluorescent sediment with P22-VLPs and supernatant with remaining monomeric protein.
Investigating Capsid Sizes
In TEM imaging we could already show separate VLPs with a diameter of around 60 nm. To analyze particle size for a greater number of capsids we used dynamic light scattering (DLS) (Fig. 7). In this method, the hydrodynamic diameter of particles can be determined. Results showed that particle sizes of in vitro assembled P22-VLPs average around 112.4 nm ± 42.31 nm. This is about 50 nm more than the expected 60 nm [4] . The distribution however shows that there is a monodisperse species of capsids. By measuring 20 randomly selected P22-VLPs we observed in the electron microscope we calculated an average diameter 57 nm ± 3 nm which is very close to the expected 60 nm.
Figure 7: Distribution of particle hydrodynamic diameters in DLS analysis. A monodisperse species of particles can be seen.
VLPs are heat stabile and exist in different conformations
It is known, that heating of P22 VLPs to 60 °C for 15 min results in a conformational change named expanded shell (EX). In the EX conformation, VLP volume is enlarged [3] . Heat treatment at 70 °C for 20 min leads to CP dissociation in distinct areas of the capsid. This results in the so-called wiffle ball (WB) conformation [3] . We performed this heat treatment with previously assembled VLPs and afterwards instantly applied these to TEM sample grids to dry them. This was done to prevent unintended re-assembly of capsids. TEM imaging revealed that the described temperature treatment did not result in the disintegration of the capsids, demonstrating their rigidity. Significant visual changes in capsid conformation that would suggest EX or WB structures were however not visible (see Fig. 8).
Figure 8: TEM images of P22-VLPs after heat treatment at a) 60°C for 15 min and b) 70°C for 20 min.
Composition of Capsids
Figure 9: SDS-PAGE of previously assembled VLPs after denaturation. Thermo Scientific PageRuler Prestained Protein Ladder was used.
For further verification, we performed SDS-PAGE of sediment we extracted carefully from the centrifuge tube. By using a rather high temperature (100 °C) for protein denaturation we expected the VLPs to mainly disintegrate into their monomers. The gel showed bands at the expected protein sizes for CP and sfGFP-SP (see Fig. 9). Since sfGFP-SP and CP have approximately the same size they can not be distinguished clearly. We know that sfGFP-SP must be integrated in the VLPs structure since the sediment in the ultracentrifuge was fluorescent.
Coat Protein builds capsids autonomously
In TEM imaging we found that CP is able to form structurally intact capsids without the presence of SP. We showed by dynamic light scattering (DLS) analysis that capsids containing only CP are smaller than P22-VLPs containing both CP and SP. This was also confirmed by measuring VLPs and CP-only capsids in TEM images using the software ImageJ (Fig. 10). Capsids which are only composed of CP (Fig. 11) measured average diameter of 53 nm ± 4.3 nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57 nm ± 3 nm (n=20; p<0.005). What also became clear is that the presence of the LPETGG tag does not affect the size of the assembled CP-only capsid.
Figure 10: Hydrodynamic diameter of CP-only capsids and unmodified VLPs.
Figure 11: Capsids containing only purified coat protein. Image made in TEM.
References
- ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [1]
- ↑ W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nat. Struct. Biol. 2003, 10, 131. [2]
- ↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [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]
