Difference between revisions of "Part:BBa K3187021"
 
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__NOTOC__
 
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<partinfo>BBa_K3187021 short</partinfo>
 
<partinfo>BBa_K3187021 short</partinfo>
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<html>
 
<html>
 +
  
 
                 <h3>Profile</h3>
 
                 <h3>Profile</h3>
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                 <tr>
 
                 <tr>
 
                 <td><b>Origin</b></td>
 
                 <td><b>Origin</b></td>
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                 <td> <i>Enterobacteria phage P22</i></td>
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                 <td> Enterobacteria phage P22</td>
 
                 </tr>
 
                 </tr>
  
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                 </table>
 
                 </table>
  
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<h3> Usage and Biology</h3>
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+
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                <h3> Usage and Biology</h3>
+
 
                  
 
                  
−
                 <p> <p>
+
                 <p>The P22 Virus-like particle&nbsp;(VLP) originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella&nbsp;typhimurium</i>.
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The P22 scaffold protein (SP) is an important part of the<i> Enterobacteria phage P22</i> 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 47kDa 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 this, 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 proteins (CP: <a href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">BBa_K3187017</a>) and 100 to 300 copies of scaffold
 +
                    proteins (SP).<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 a mixture of monomers and dimers is 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 CPs rearrange, 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 SPs and CPs, but it also can assemble with only CPs. If it assembles without SPs it can form into
 +
                    two different 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&nbsp;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">
−
                             <a href="#cite_note-1">[1]
+
                             <a href="#cite_note-1">[7]
 
                             </a>  
 
                             </a>  
 
                     </sup>  
 
                     </sup>  
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<sup id="cite_ref-2" class="reference">
 
<sup id="cite_ref-2" class="reference">
−
                             <a href="#cite_note-2">[2]
+
                             <a href="#cite_note-2">[3]
 
                             </a>  
 
                             </a>  
−
                     </sup>.
+
                     </sup>
 
<br>  
 
<br>  
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  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  
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  After the assembly of the virus&#8209;capsid the SPs are released into the capsid. In case of a functional P22 bacteriophage, this protein is  
 +
extracted out of the capsid <i>in&nbsp;vivo</i> while the viral DNA is loaded into the capsid.
  
  
 
<sup id="cite_ref-3" class="reference">
 
<sup id="cite_ref-3" class="reference">
−
                             <a href="#cite_note-3">[3]
+
                             <a href="#cite_note-3">[8]
 
                             </a>  
 
                             </a>  
 
                     </sup>  
 
                     </sup>  
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<sup id="cite_ref-4" class="reference">
 
<sup id="cite_ref-4" class="reference">
−
                             <a href="#cite_note-4">[4]
+
                             <a href="#cite_note-4">[9]
 
                             </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.
  
  
 
<sup id="cite_ref-5" class="reference">
 
<sup id="cite_ref-5" class="reference">
−
                             <a href="#cite_note-5">[5]
+
                             <a href="#cite_note-5">[10]
 
                             </a>  
 
                             </a>  
 
                     </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&#8209;Terminus of the SP, as the C&#8209;Terminus is important for mechanism of the assembly.
  
  
 
<sup id="cite_ref-6" class="reference">
 
<sup id="cite_ref-6" class="reference">
−
                             <a href="#cite_note-6">[6]
+
                             <a href="#cite_note-6">[11]
 
                             </a>  
 
                             </a>  
 
                     </sup>  
 
                     </sup>  
  
−
.
+
 
 
                 </p>
 
                 </p>
−
               
+
             
−
 
+
−
 
+
−
 
+
 
                 <h3> Methods</h3>
 
                 <h3> Methods</h3>
−
             
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                 <h4>Cloning</h4>
−
                 <h4>Assembly</h4>
+
                 <p>The fusion protein sfGFP&#8209;SP (<a href="https://parts.igem.org/Part:BBa_K3187003" target="_blank">BBa_K3187003</a>) was cloned into the pACYCT2 backbone via <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">Gibson Assembly</a>. The sfGFP was then deleted using restriction digest.</p>
−
                 <p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with
+
−
                    ultracentrifugation  <a href="#"target="_blank">ultracentrifugatione</a> and are visualized with
+
−
                    <a href="#"target="_blank">TEM</a>. For more information look at our <a href="#"target="_blank">wiki</a>
+
−
             
+
  
−
                 <h3>Results</h3>  
+
                 <h4>Purification</h4>
 +
                <p>The protein was heterologously expressed in <i>E.&nbsp;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&#8209;tag&nbsp;II.
 +
                </p>
  
−
               
+
                 <h4>SDS-PAGE and western blot</h4>
−
 
+
                 <p>To verify that the CP&#8209;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
−
                 <h4> Assembly</h4>
+
                    <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">western blot</a>, was performed.
−
                 <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="#"target="_blank">wiki</a>.
+
−
                        <div style="text-align: center;"> 
+
−
                        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png" style="max-width:60%" />
+
−
                          <div class="caption">
+
−
                          <p>
+
−
                          <b>Figure 2:</b> Ultracentrifugation of assembled VLPs
+
−
                               
+
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                            </p>
+
−
                          </div>
+
−
                        </div>
+
 
                 </p>
 
                 </p>
−
                <p> The images of TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. As a result,
 
−
                    the CPs produced using this part are fully functional . The CPs assemble with
 
−
                    scaffold proteins (SPs) and they can be modified on the surface (Fig. 4). Moreover, CPs also assemble without SPs
 
−
                    (Fig. 3).
 
−
                </p>
 
−
                <div style="text-align: center;"> 
 
  
−
                    <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg" style="max-width:40%" />
+
                <h3>Results</h3>
−
                     
+
                <h4>Cloning and Expression</h4>
 +
                <p>The successful cloning was verified with Sanger sequencing and production was verified with a western blot.
 +
                        <div> 
 +
                        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/1/1a/T--TU_Darmstadt--westernplot_sp.jpeg" style="max-width:40%" />
 
                         <div class="caption">
 
                         <div class="caption">
 
                           <p>
 
                           <p>
−
                           <b>Figure 3:</b>
+
                           <b>Figure 1:</b>
−
                                 Assembly of only coat proteins with LPETGG.
+
                                 Western blot of all produced and purified proteins.  
 
                             </p>
 
                             </p>
 
                         </div>
 
                         </div>
−
                </div>
 
−
                    <p>Fig. 3 shows that no scaffold proteins are necessary for assembly.</p>   
 
−
                   
 
−
                    <div style="text-align: center;"> 
 
−
                    <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg" style="max-width:40%" />
 
−
                       
 
−
                        <div class="caption">
 
−
                          <p>
 
−
                          <b>Figure 4:</b>
 
−
                                Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
 
−
                            </p>
 
 
                         </div>
 
                         </div>
−
                    </div>
+
                <p><b>Fig. 1</b> shows that sfGFP-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 the upper band scaffold protein. We came to this conclusion by comparing the lane of sfGFP&#8209;SP with lanes of only SP, and SP 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.
−
                   
+
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 had been verified by sequencing, 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 unexpectedly in this SDS&#8209;PAGE.
−
                    <p>Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and CP-LPETGG can be modified for this process</p>   
+
−
 
+
−
 
+
−
 
+
−
                    <h2>References</h2>
+
  
 +
                  </p> 
 +
<p>
 +
For more information about VLP assembly,
 +
                        visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP" target="_blank">wiki</a>.     
  
 +
                    <h2>References</h2>
 
                     <ol class="references">
 
                     <ol class="references">
 
                         <li id="cite_note-1">
 
                         <li id="cite_note-1">
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                             </span>
 
                             </span>
 
                             <span class="reference-text">
 
                             <span class="reference-text">
−
                            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.
+
                            Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics,
−
                             <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1016/0022-2836(76)90278-3">[1] </a>
+
                            Structure, and Mechanism, 2005, pp 80- 88
 +
                             <a rel="nofollow" class="external autonumber" href="https://link.springer.com/chapter/10.1007/0-387-28521-0_5">[1] </a>
 
                             </span>
 
                             </span>
 
                         </li>
 
                         </li>
−
 
−
 
 
                      
 
                      
 
                         <li id="cite_note-2">
 
                         <li id="cite_note-2">
Line 170: Line 172:
 
                             </span>
 
                             </span>
 
                             <span class="reference-text">
 
                             <span class="reference-text">
−
                                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.
+
                            Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP,
−
                             <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1038/nsb891">[2] </a>
+
                            Chemical Communications, 2013, 49: 10412-10414
 +
                             <a rel="nofollow" class="external autonumber" href="https://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc46517a#!divAbstract">[2] </a>
 
                             </span>
 
                             </span>
 
                         </li>
 
                         </li>
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                             <span class="mw-cite-backlink">
 
                             <span class="mw-cite-backlink">
 
                                 <a href="#cite_ref-3">↑</a>
 
                                 <a href="#cite_ref-3">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                            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     
 +
                            <a rel="nofollow" class="external autonumber" href="https://www.nature.com/articles/nsb891">[3] </a>
 +
                            </span>
 +
                        </li>
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                            Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein,
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                            Journal of Molecular Biology, 1998, Volume 281: 69-79     
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                            <a rel="nofollow" class="external autonumber" href="https://www.sciencedirect.com/science/article/pii/S0022283698919179">[4] </a>
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                            Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid
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                            of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 
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                            P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size
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                            determination suggested by T = 4 and T = 7 procapsid structures.,Biophysical Journal, 1998, 74: 559-568     
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                            <a rel="nofollow" class="external autonumber" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299408/">[6] </a>
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                            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.
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                               King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119.   
 
                               King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119.   
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                                 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.  
 
                                 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.  
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                                 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
 
                                 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  
 
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                                 P. R. Weigele, L. Sampson, D. Winn‐Stapley, S. R. Casjens, Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains
 
                                 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.  
 
, J. Mol. Biol. 2005, 348, 831.  
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Latest revision as of 17:35, 21 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 Virus-like particle (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 this, and its specific properties, it generates an accessible VLP platform. [1]

An assembled P22 VLP consists of 420 copies of coat proteins (CP: BBa_K3187017) and 100 to 300 copies of scaffold proteins (SP). [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 a mixture of monomers and dimers is 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 CPs rearrange, 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 SPs and CPs, but it also can assemble with only CPs. If it assembles without SPs it can form into two different 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. [7] [3]
After the assembly of the virus‑capsid the SPs are 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. [8] [9] 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. [10] This fusion has to occur at the N‑Terminus of the SP, as the C‑Terminus is important for mechanism of the assembly. [11]

Methods

Cloning

The fusion protein sfGFP‑SP (BBa_K3187003) was cloned into the pACYCT2 backbone via Gibson Assembly. The sfGFP was then deleted using restriction digest.

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.

Results

Cloning and Expression

The successful cloning was verified with Sanger sequencing and production was verified with a western blot.

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

Fig. 1 shows that sfGFP-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 the upper band scaffold protein. We came to this conclusion by comparing the lane of sfGFP‑SP with lanes of only SP, and SP 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 had been verified by sequencing, 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 unexpectedly in this SDS‑PAGE.

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. ↑ King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. [8]
  9. ↑ 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. [9]
  10. ↑ 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 [10]
  11. ↑ 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. [11]


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 351
  • 1000
    COMPATIBLE WITH RFC[1000]