Difference between revisions of "Part:BBa K3187002"

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P22 Bacteriophage Scaffolding Protein

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Usage and Biology

The P22 scaffold protein (SP) is an important part of the Enterobacteria phage P22 capsid. The virus capsid is assembled with the help of up to 300 copies of the 18 kDa scaffold protein out of approx. 400 copies of the 47 kDa coat protein ^{[1] [2]} .
After the assembly of the virus-capsid the SP is released into the capsid. In case of a functional P22 bacteriophage, this protein is extracted out of the capsid in vivo while the viral DNA is loaded into the capsid ^{[3] [4]} . Because the artificial capsid is not filled with DNA the SP remains in the capsid. By fusing the SP with a cargo-protein, one can load the capsid with said cargo ^[5] . This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly ^[6] .
Here the scaffold protein(SP) is fused with a Strep-tagII for protein purification. The construct contains a pT7 promoter for the T7 polymerase, a lac operator, so expression can be induced with IPTG, and T7 Terminator.

Methods

Cloning

This constructed was cloned using PCR with overhang primers and restriction and ligation out of sfGFP-SP construct BBa_K3187003. To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The SP was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure FPLC.Strep-tag II was used as affinity tag.

SDS-Page and Western blot

To verify that the Protein was produced, a SDS-Page SDS-Page followed by a Western blot was performed.

Assembly

The assembly was tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation ultracentrifugatione and are visualized with TEM.

Results

Cloning and Expression

The successful cloning was proven with sanger sequencing and production with a Western blot.

Figure 1: Western blot of all produced and purified proteins.

Fig. 1 shows that SP has a molecular weight of approximatley 30 kDa. This is more than the theoretical weight. Because the fusion prtoein of SP and sfGFP (BBa_K3187003) has the expected length and has two more bands that have the length of sfGFP and of SP and because the sequencing wethink that we haeve the right proein but it behaves unexpected in the SDS-Page. The proteins were detected with Strep-Tactin-HRP.

Assembly

The images of ultracentrifugation displays 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 reatment, 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 of 60 nm or less (Fig. 2). For more information about VLP assembly, visit our wiki.

Figure 2: TEM picture of assembled VLPs

References

1. ↑ 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]
2. ↑ 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]
3. ↑ King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. [3]
4. ↑ S. Casjens and R. Hendrix, (1988) "Control mechanisms in dsDNA bacteriophage assembly", in The Bacteriophages, volume 1, ed. R. Calendar, Plenum Press, p. 15-91. [4]
5. ↑ Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas, Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle ,ACS Chemical Biology 2014 9 (2), 359-365 [5]
6. ↑ P. R. Weigele, L. Sampson, D. Winn‐Stapley, S. R. Casjens, Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains , J. Mol. Biol. 2005, 348, 831. [6]

Sequence and Features