Difference between revisions of "Part:BBa K3187021"

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	<h3>Profile</h3>		<h3>Profile</h3>
−	<table style=“width:80%“>	+	<table style="width:80%">
	<tr>		<tr>
	<td><b>Name</b></td>		<td><b>Name</b></td>
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	<h3> Usage and Biology</h3>		<h3> Usage and Biology</h3>
−	<p> <p>	+	<p>The P22 VLP originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella&nbsp;typhimurium</i>.
−	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&nbsp;kDa coat protein	+	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.<sup id="cite_ref-1" class="reference">
		+	<a href="#cite_note-1">[1]</a></sup><br>
		+	</p>
		+	<p>An assembled P22 VLP consists of 420&nbsp;copies of coat protein (CP: <a href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">BBa_K3187017</a>) and 100 to 300 copies of scaffold
		+	protein (SP: <a href="https://parts.igem.org/Part:BBa_K3187021" target="_blank">BBa_K3187021</a>).<sup id="cite_ref-2" class="reference">
		+	<a href="#cite_note-2">[2]</a>
		+	</sup><br>
		+	The shell of the VLP is formed by the 46.6&nbsp;kDa&nbsp;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.<sup id="cite_ref-3" class="reference">
		+	<a href="#cite_note-3">[3]</a>
		+	</sup><br>
		+	The 18&nbsp;kDa&nbsp;SP is required for an efficient assembly and naturally consists of 303&nbsp;amino acids. It has been shown, that an
		+	N&#8209;terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D&#8209;structure is composed of segmented helical
		+	domains, with little or no globular core. In solution is a mixture of monomers and dimers present.<sup id="cite_ref-4"
		+	class="reference">
		+	<a href="#cite_note-4">[4]</a>
		+	</sup>
		+	When purified CPs and SPs are mixed, they self&#8209;assemble into VLPs. </p>
		+
		+	<p> P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60&nbsp;°C, the CP rearranges, forming
		+	the expanded shell form&nbsp;(EX). This form has a diameter of about 58&nbsp;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 &nbsp;°C. The
		+	whiffleball has 10&nbsp;nm pores, while the procapsid or the expanded shell form only have 2&nbsp;nm pores.<sup id="cite_ref-5" class="reference">
		+	<a href="#cite_note-5">[5]</a>
		+	</sup>
		+	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&nbsp;=&nbsp;4 icosahedral lattice with a diameter between 195&nbsp;Å and 240&nbsp;Å. The
		+	larger capsid also has an icosahedral lattice, but it is formed as T&nbsp;=&nbsp;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&nbsp;Å and 306&nbsp;Å. Each capsid consists of a 85&nbsp;Å thick icosahedral shell made of CP.<sup id="cite_ref-6" class="reference">
		+	<a href="#cite_note-6">[6] </a>
		+	</sup></p>
		+	<p> <p>
		+	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&nbsp;kDa coat protein.
	<sup id="cite_ref-1" class="reference">		<sup id="cite_ref-1" class="reference">
Line 50:		Line 87:
	<a href="#cite_note-2">[2]		<a href="#cite_note-2">[2]
	</a>		</a>
−	</sup>.	+	</sup>
	<br>		<br>
−	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 <i>in vivo</i> while the viral DNA is loaded into the capsid	+	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 <i>in vivo</i> while the viral DNA is loaded into the capsid.
Line 64:		Line 102:
	<a href="#cite_note-4">[4]		<a href="#cite_note-4">[4]
	</a>		</a>
−	</sup> . 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	+	</sup> 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.
Line 72:		Line 111:
	</sup>		</sup>
−	. This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly	+	This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly.
Line 80:		Line 119:
	</sup>		</sup>
−	.	+
	</p>		</p>
Line 88:		Line 127:
	<h3> Methods</h3>		<h3> Methods</h3>
−	<h4>Assembly</h4>	+
−	<p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with	+
−	ultracentrifugation  <a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">ultracentrifugatione</a> and are visualized with	+
−	<a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">TEM</a>.	+
Line 98:		Line 134:
−	<h4> Assembly</h4>	+
−	<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	+
−	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 <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"target="_blank">wiki</a>.	+
−		+
−		+
−	<div style="text-align: center;">	+
−	<img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/f/fa/T--TU_Darmstadt--TEM_SP_without_sfGFP.png" style="max-width:60%" >	+
−	<div class="caption">	+
−	<p>	+
−	<b>Figure 1:</b> TEM picture of assembled VLPs	+
−		+
−	</p>	+
−	</div>	+
−	</div>	+

---

Revision as of 18:22, 20 October 2019

P22 Bacteriophage Scaffolding Protein

Profile

Name	Scaffold protein
Base pairs	489
Molecular weight	18 kDa
Origin	Enterobacteria phage P22
Properties	In combination with the coat protein (BBa_K3187017) this protein builds the virus capsid of the P22 phage.

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]

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]

Methods

Results

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