Of course there are more parts necessary in order to express the CP‑LPETGG heterologously in E. coli BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is transcribed into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the protein. The T7 promoter (BBa_K3187029) is recognized by the T7 polymerase. In order to regulate the protein production, the lac‑operator (BBa_K3187029) was used. Furthermore, a RBS (BBa_K3187029) is in the construct and a Short Linker (5AA) (BBa_K3187030) is found between CP and LPETGG. The T7 terminator (BBa_K3187032) and Strep-tag II (BBa_K3187025) are located downstream of the coat protein CDS.

Methods

Cloning

The CP-LPETGG was cloned into the pET24-backbone with restriction and ligation . To do this, the CP‑LPETGG, as well as the T7 promoter and the lac‑operator sequence, was ordered from Integrated DNA Technologies (IDT). To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The CP‑LPETGG was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-tag II.

SDS-PAGE and western blot

To verify that the CP-LPETGG was produced, a SDS-PAGE followed by a western blot was performed.

Sortase-mediated ligation

In order to characterize CP‑LPETGG, different assays were performed. The possibility of modifying the CP was tested with mCherry and Sortase A7M. The Sortase A7M successfully linked mCherry and CP‑LPETGG. The linkage was verified with a SDS‑PAGE. To identify whether the Sortase A7M or Sortase A5M produce multimers of coat proteins with LPETGG‑tag, CP‑LPETGG and Sortase A7M and Sortase A5M were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS‑PAGE.

Assembly

The assembly was tested in vivo and in vitro. The assembled VLPs were collected with ultracentrifugation and were visualized with transmission electron microscopy (TEM). Therefore, the in vivo assembled VLPs are purified with size-exclusion chromatography (SEC) (Sephadex-100 column) ^[9] The diameter of VLPs was measured with dynamic light scattering (DLS) analysis.

Results

Cloning and Expression

The successful cloning was confirmed with sanger sequencing. The purification was documented with an chromatogram and the successful production of the VLPs was confirmed with a western blot.

The chromatogram shows a peak for elution between 52 mL and 56 mL. The maximum is found approximatley at 380 mAU (Fig. 2 and 3).

Fig. 4 shows western blot of produced and purified proteins. CP‑LPETGG was detected using a Strep‑Tactin‑HRP conjugate. CP‑LPETGG can be seen at approximately 49 kDa. Consequently, the successful production was proven.

Sortase-mediated ligation

Want to know more about sortase-mediated ligation? Please have a look at our wiki.

In order to test the funtion of the genetically fused sortase-tag LPETGG to the CP, we performed the linking reaction with CP-LPETGG and GGGG-mCherry. Resulting protein mix was applied to a SDS-PAGE. We saw products at the expected size (28 kDa + 49 kDa = 77 kDa) thus the requirement is fulfilled. However, a lot of additional bands appeared that we did not expect. These bands also appeared when only Sortase A7M and CP were mixed.

Figure 5:

a) Sortase A7M band is at expected height (17.85 kDa). The two negative controls containing only GGGG-mCherry (28 kDa) and CP-LPETGG (49 kDa) at the expected respective heights. b) Shown are sfGFP-SP and CP-LPETGG each incubated with both Sortase A7M and Sortase A5M. Both gels display multimers when coat and a sortase variant are in a sample together.

To investigate this issue, we had a look at the literature and found a matching description in the publication of Patterson et al.. They performed a similar experiment with P22 capsid proteins and observed the same multimers in their SDS-PAGEs . Comparing both SDS-PAGEs, we came to the following assumption:

Because of the promiscuity of Sortase A7M to accept primary amines as substrates, as we discussed previously, the formation of CP multimers occurs, unspecifically catalyzed by Sortase A7M.

Parallel to these experiments, we successfully modified the exterior of pre-assembled VLPs in vitro (VLP assembly). These modified VLPs were homogenous and overall correctly assembled. Therefore, we conclude that the described multimer problem only occurs when Sortase A7M encounters free CP.

Assembly

Ultracentrifugation was used to harvest VLPs consisting of CP (BBa_K3187017) and SP after in vivo and in vitro assembly

In vivo assembled VLPs

For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG, 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 with a sucrose cushion (35% w/v) at 150,000 x g was used as a first concentration step. The resulting sediment contained fluorescent material which we suspected to contain a concentrated fraction of VLPs.

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. For getting rid of these contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex‑100 column). ^[12] After SEC the elution sample with the highest suspected VLP concentration (based on UV absorption) was imaged with transmission electron microscopy (TEM). Numerous capsids in the correct size range were clearly visible. 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 this ^[12] suggests.

In vitro assembled VLPs

The images of ultracentrifugation show that monomeric proteins were separated from assembled capsids by ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation treatment, a sediment was clearly visible in the centrifuge tube, which we suspected to mainly contain VLPs. 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 of 60 nm or less (Fig. 8).

The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, the CPs produced using this part must be fully functional. The CPs assemble with SPs and can be modified on the surface (Fig. 8). Moreover, CPs also assemble without SPs (Fig.  9).

Fig. 9 shows that no scaffold proteins are necessary for assembly.

Fig. 10 shows that CP-LPETGG and SPs assemble to VLPs with structural integrity and that CP-LPETGG can be modified for this process.

For more information about VLP assembly, please visit our wiki.

Sortase-mediated ligation of GGGG-tagged proteins to the surface of the assembled P22 VLP

Ultracentrifugation

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. 11). This is a strong indication that sortase has attached sfGFP to the VLP exterior, as only assembled VLPs accumulate in the sediment. ^[8] We then prepared the ultracentrifugation sediment for transmission electron microscopy. Encouragingly, we observed numerous visually intact VLPs.