Difference between revisions of "Part:BBa K3187003"
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| − | < | + | <h1>Profile</h1> |
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| − | < | + | <h1> Usage and Biology</h1> |
<p>The P22 VLP originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella typhimurium</i>. | <p>The P22 VLP originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella typhimurium</i>. | ||
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</p> | </p> | ||
| − | < | + | <h1> Methods</h1> |
| − | < | + | <h2>Cloning</h2> |
<p>The fusion protein was cloned into the pACYCT2 backbone <i>via</i> <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">Gibson Assembly</a>. To verify the cloning, | <p>The fusion protein was cloned into the pACYCT2 backbone <i>via</i> <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">Gibson Assembly</a>. To verify the cloning, | ||
the sequence was controlled by Sanger sequencing by Microsynth Seqlab. | the sequence was controlled by Sanger sequencing by Microsynth Seqlab. | ||
</p> | </p> | ||
| − | < | + | <h2>Purification</h2> |
<p>The protein was heterologously expressed in <i>E. coli</i> BL21 and purified with | <p>The protein was heterologously expressed in <i>E. coli</i> BL21 and purified with | ||
<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">GE Healthcare ÄKTA FPLC</a>. The used affinity tag was Strep‑tag II. | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">GE Healthcare ÄKTA FPLC</a>. The used affinity tag was Strep‑tag II. | ||
</p> | </p> | ||
| − | < | + | <h2>SDS-PAGE and western blot</h2> |
<p>To verify that the CP‑LPETGG was produced, a <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SDS-PAGE</a> followed by a | <p>To verify that the CP‑LPETGG was produced, a <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SDS-PAGE</a> followed by a | ||
<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">western blot</a> was performed. | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">western blot</a> was performed. | ||
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| − | < | + | <h2>Assembly</h2> |
<p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with | <p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with | ||
<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">ultracentrifugation</a> and are visualized with | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">ultracentrifugation</a> and are visualized with | ||
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| − | < | + | <h1>Results</h2> |
| − | < | + | <h2>Cloning and Expression</h2> |
<p>The successful cloning was proven with Sanger sequencing and production with a western blot. | <p>The successful cloning was proven with Sanger sequencing and production with a western blot. | ||
<div> | <div> | ||
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| − | < | + | <h2><i>In vitro</i> Assembly</h2> |
<p> The images of ultracentrifugation displays that monomeric proteins were separated from assembled capsids by | <p> 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 | ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation | ||
Revision as of 11:42, 21 October 2019
Superfolder Green Fluorescence Protein x P22 Bacteriophage Scaffolding Protein Fusion
Profile
| Name | sfGFP scaffold protein fusion protein |
| Base pairs | 1547 |
| Molecular weight | 46.1 kDa |
| Origin | Enterobacteria phage P22; Aequorea victoria |
| Parts | T7-Promoter, lac-operator, RBS(g10 leader sequence), sfGFP, Strep‑tag II, scaffold protein (SP), double terminator (rrnB T1 terminator and T7Te terminator) |
| Properties | P22 capsid assembly; loading the capsid with sfGFP |
Usage and Biology
The P22 VLP originates from the temperate bacteriophage P22. Its natural host is Salmonella typhimurium.
Since it was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for temperate phages.
To date, nearly everything is known about its lifecycle. Because of that and its specific properties it generates
an accessible VLP platform.
[1]
An assembled P22 VLP consists of 420 copies of coat protein (CP: BBa_K3187017) and 100 to 300 copies of scaffold
protein (SP: BBa_K3187021).
[2]
The shell of the VLP is formed by the 46.6 kDa CP. The coat protein occurs in one configuration, which contains a globular
structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric units, which form
the icosahedral structure of the VLP.
[3]
The 18 kDa SP is required for an efficient assembly and naturally consists of 303 amino acids. It has been shown, that an
N‑terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D‑structure is composed of segmented helical
domains, with little or no globular core. In solution is a mixture of monomers and dimers present.
[4]
When purified CPs and SPs are mixed, they self‑assemble into VLPs.
P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60 °C, the CP rearranges, forming the expanded shell form (EX). This form has a diameter of about 58 nm and the volume is doubled compared to the one of the procapsid. The expanded shell form changes into the whiffleball form (WB) when heated further up to 70 °C. The whiffleball has 10 nm pores, while the procapsid or the expanded shell form only have 2 nm pores. [5] Furthermore, the P22 VLP consists of SP and CP, but it also can assemble with only CPs. If it assembles without SP it can form two sizes of capsids. The small capsid is built as a T = 4 icosahedral lattice with a diameter between 195 Å and 240 Å. The larger capsid also has an icosahedral lattice, but it is formed as T = 7. T being the "triangulation number", a measure for capsid size and complexity. Moreover, it is like the wild type VLP, which includes the SP. The diameter of the wild type VLP, is between 260 Å and 306 Å. Each capsid consists of a 85 Å thick icosahedral shell made of CP. [6]
This protein is a fusion protein consisting of the GFP variant sfGFP (superfolder GFP: BBa_K3187022) and the scaffold protein from Enterobacteria phage P22, which is one of the two proteins needed for the assembly of the virus capsid [7] [8] . By fusing these two proteins we can load our VLPs with sfGFP [9] . In between those two proteins is a Strep‑tag II for protein purification.The protein expression is induced with isopropyl β‑d‑1‑thiogalactopyranoside, starting the expression of the T7 polymerase in E. coli BL21(DE3), that binds to the T7 promoter and removing the repressor from the lac‑operator.
Methods
Cloning
The fusion protein was cloned into the pACYCT2 backbone via Gibson Assembly. To verify the cloning, the sequence was controlled by Sanger sequencing by Microsynth Seqlab.
Purification
The protein was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA 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.
Assembly
The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation 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 45 kDa. This is about the expected size of 46.1 kDa. Two additional bands in this lane 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 a Strep‑Tactin‑HRP western blot. The band of Strep‑tag II 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.
In vitro 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 treatment, 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).
Figure 2: Ultracentrifugation of assembled VLPs
Fig.2 shows assembled VLPs taken via TEM. The green flourescence of the VLP pellet indicates that we succesfully loaded our VLPs with sfGFP by using our sfGFP-SP fusion protein.
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 ImageJ. Capsids which are only composed of CP 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 3: Diagram of DLS measurment of VLPs .
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. 5). Moreover, CPs also assemble without SPs (Fig. = 4).
Figure 4:
Assembly of only coat proteins with a LPETGG-tag.
It also shows that no scaffold proteins are necessary for assembly.
Figure 5: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
Our expectations that VLPs, not containing the scaffold protein, are less intact and more unstable (Fig. 4) compared to the ones including a scaffold (Fig. 5) were confirmed.
For more information about VLP assembly, visit our wiki.
References
- ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
- ↑ Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions,Nature Structural Biology, 2003, 10: 131-135 [3]
- ↑ Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69-79 [4]
- ↑ Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 [5]
- ↑ P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.,Biophysical Journal, 1998, 74: 559-568 [6]
- ↑ 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. [7]
- ↑ 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. [8]
- ↑ 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 [9]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 807
Illegal XhoI site found at 1338 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1196
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1384
Illegal SapI.rc site found at 105