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−	. In between those two proteins is a Strep&#8209;tag&nbsp;II for protein purification.	+	. In between those two proteins is a <a href="https://parts.igem.org/Part:BBa_K3187025" target="_blank">Strep&#8209;tag&nbsp;II</a> for protein purification.
	<br>		<br>
	The protein expression is induced with isopropyl β&#8209;d&#8209;1&#8209;thiogalactopyranoside, starting the expression of the T7 polymerase in <i>E.&nbsp;coli</i>		The protein expression is induced with isopropyl β&#8209;d&#8209;1&#8209;thiogalactopyranoside, starting the expression of the T7 polymerase in <i>E.&nbsp;coli</i>
−	BL21(DE3), that binds to the T7&nbsp;promoter and removing the repressor from the <i>lac</i>&#8209;operator.</p>	+	BL21(DE3), that binds to the T7&nbsp;promoter <a href="https://parts.igem.org/Part:BBa_K31870029" target="_blank">(BBa_K31870029)</a>and removing the repressor from the <i>lac</i>&#8209;operator <a href="https://parts.igem.org/Part:BBa_K31870029" target="_blank">(BBa_K31870029)</a>.</p>
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−	<p><b>Fig. 1</b> shows that SP has a molecular weight of approximatley 45&nbsp;kDa. This is about the expected size of 46.1&nbsp;kDa. Two additional bands in this lane can be observed. One at est.&nbsp;25&nbsp;kDa and one between 25 and 37&nbsp;kDa. The lower band may be sfGFP and upper band scaffold protein. We came to this conclusion by comparing the lane of sfGFP&#8209;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&#8209;SP fusion protein. During denaturation for SDS&#8209;PAGE sample preparation, the fusion protein can break in two parts, sometimes it breaks in front and sometimes after the Strep&#8209;tag&nbsp;II. This is indicated by the fact that both bands are stained by a Strep&#8209;Tactin&#8209;HRP western blot.	+	<p><b>Fig. 1</b> shows that SP has a molecular weight of approximatley 45&nbsp;kDa. This is about the expected size of 46.1&nbsp;kDa. Two additional bands in this lane can be observed. One at est.&nbsp;25&nbsp;kDa and one between 25 and 37&nbsp;kDa. The lower band may be sfGFP and upper band scaffold protein. We came to this conclusion by comparing the lane of sfGFP&#8209;SP with lanes of only SP and with sfGFP with TEV cleavage site <a href="https://parts.igem.org/Part:BBa_K3187004)" target="_blank">(BBa_K3187004</a>. Those two bands are probably produced by the denaturing of the sfGFP&#8209;SP fusion protein. During denaturation for SDS&#8209;PAGE sample preparation, the fusion protein can break in two parts, sometimes it breaks in front and sometimes after the Strep&#8209;tag&nbsp;II. This is indicated by the fact that both bands are stained by a Strep&#8209;Tactin&#8209;HRP western blot.
	The band of Strep&#8209;tag&nbsp;II and SP can be observed at a size of est.&nbsp;30&nbsp;kDa. This is larger than the expected, theoretical size of SP at about 18&nbsp;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&#8209;PAGE.		The band of Strep&#8209;tag&nbsp;II and SP can be observed at a size of est.&nbsp;30&nbsp;kDa. This is larger than the expected, theoretical size of SP at about 18&nbsp;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&#8209;PAGE.
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	<h2><i>In vivo assembled VLPs</i></h2>		<h2><i>In vivo assembled VLPs</i></h2>
−	<p>For extracting the VLPs, which consits of SP and with sfGFP modified CP&#8209;LPETGG, directly from cell broth we first	+	<p>For extracting the VLPs, which consits of SP and with sfGFP modified CP&#8209;LPETGG
		+	<a href="https://parts.igem.org/Part:BBa_K3187000" target="_blank">(BBa_K3187000)</a>, directly from cell broth we first
	lysed the cells by sonication and got rid of debris by two		lysed the cells by sonication and got rid of debris by two
	centrifugation steps at 12,000&nbsp;x&nbsp;g. Afterwards ultracentrifugation with a sucrose cushion (35%&nbsp;w/v) at 150,000&nbsp;x&nbsp;g was		centrifugation steps at 12,000&nbsp;x&nbsp;g. Afterwards ultracentrifugation with a sucrose cushion (35%&nbsp;w/v) at 150,000&nbsp;x&nbsp;g was
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	the SPs produced using this part must be fully functional. The CPs assemble with		the SPs produced using this part must be fully functional. The CPs assemble with
	SPs and can be modified on the surface (<b>Fig.&nbsp;5</b>). Moreover, CPs also assemble without SPs		SPs and can be modified on the surface (<b>Fig.&nbsp;5</b>). Moreover, CPs also assemble without SPs
−	(<b>Fig. =&nbsp;4</b>).	+	(<b>Fig.&nbsp;4</b>).
	</p><br>		</p><br>
	<div>		<div>

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Revision as of 17:30, 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]

The scaffold 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 (BBa_K31870029)and removing the repressor from the lac‑operator (BBa_K31870029).

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.

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 (BBa_K3187004. 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 vivo assembled VLPs

For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG (BBa_K3187000), 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 ^[12] suggest.

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).

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.

The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, the SPs 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).

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

1. ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
2. ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
3. ↑ 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]
4. ↑ Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69-79 [4]
5. ↑ 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]
6. ↑ 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]
7. ↑ 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]
8. ↑ 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]
9. ↑ 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