Difference between revisions of "Part:BBa K3187002"
 
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__NOTOC__
 
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<partinfo>BBa_K3187002 short</partinfo>
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<partinfo>BBa_K3187021 short</partinfo>
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<html>
 
<html>
  
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<h3>Profile</h3>
 
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<table style=“width:80%“>
 
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    <tr>
 
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        <td><b>Name</b></td>
 
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        <td>P22 Bacteriophage Scaffolding Protein</td>
 
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    </tr>
 
  
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    <tr>
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                <h3>Profile</h3>
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        <td><b>Base pairs</b></td>
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                <table style="width:80%">
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        <td>782</td>
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                <tr>
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    </tr>
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                <td><b>Name</b></td>
 +
                <td>Scaffold protein </td>
 +
                </tr>
  
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    <tr>
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                <tr>
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        <td><b>Molecular weight</b></td>
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                <td><b>Base pairs</b></td>
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        <td>19.3 kDa</td>
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                <td> 782</td>
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    </tr>
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                </tr>
  
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    <tr>
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                <tr>
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        <td><b>Origin</b></td>
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                <td><b>Molecular weight</b></td>
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        <td> <i>Enterobacteria phage P22</i></td>
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                <td>18 kDa</td>
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    </tr>
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                </tr>
  
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    <tr>
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                <tr>
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        <td><b>Parts</b></td>
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                <td><b>Origin</b></td>
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        <td> pT7-promoter, lac Operator, Strep-tag II, Scaffold protein,T7 Terminator </td>
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                <td> Enterobacteria phage P22</td>
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    </tr>
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                 </tr>
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    <tr>
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        <td><b>Properties</b></td>
+
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        <td> In combination with the coat protein<a href=”https://parts.igem.org/Part:BBa_K3187001”>(BBa_K3187001) </a>
+
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                 this protein builds the virus capsid of the P22 phage. </td> </tr> </table> <h3> Usage and Biology</h3>
+
  
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                 <p>
+
           
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                    The P22 scaffold protein (SP) is an important part of the Enterobacteria phage P22 capsid. The virus
+
                 <tr>
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                    capsid is assembled with the help of up to 300 copies of the 18&nbsp;kDa scaffold protein out of
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                <td><b>Properties</b></td>
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                    approx. 400 copies of the 47&nbsp;kDa coat protein
+
                <td> In combination with the coat protein <a href="https://parts.igem.org/Part:BBa_K3187017">(BBa_K3187017)</a> this protein builds the virus capsid of the P22 phage. </td>
−
                    <sup id="cite_ref-1" class="reference">
+
                </tr>
−
                        <a href="#cite_note-1">[1]
+
                </table>
−
                        </a>
+
−
                    </sup>
+
  
  
−
                    <sup id="cite_ref-2" class="reference">
 
−
                        <a href="#cite_note-2">[2]
 
−
                        </a>
 
−
                    </sup>
 
−
                    .
 
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                    <br>
 
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                    After the assembly of the virus-capsid the SP is released into the capsid. In case of a functional
 
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                    P22 bacteriophage, this protein is extracted out of the capsid <i>in vivo</i> while the viral DNA is
 
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                    loaded into the capsid
 
  
−
                     <sup id="cite_ref-3" class="reference">
+
                <h3> Usage and Biology</h3>
−
                        <a href="#cite_note-3">[3]
+
               
−
                        </a>
+
                <p>The P22 VLP originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella&nbsp;typhimurium</i>.
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                     </sup>
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                    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).<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">
 +
                            <a href="#cite_note-1">[7]
 +
                            </a>
 +
                    </sup>
  
−
                    <sup id="cite_ref-4" class="reference">
 
−
                        <a href="#cite_note-4">[4]
 
−
                        </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 id="cite_ref-5" class="reference">
+
<sup id="cite_ref-2" class="reference">
−
                        <a href="#cite_note-5">[5]
+
                            <a href="#cite_note-2">[3]
−
                        </a>
+
                            </a>  
 
                     </sup>
 
                     </sup>
 +
<br>
 +
After the assembly of the virus&#8209;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&nbsp;vivo</i> while the viral DNA is loaded into the capsid.
  
  
−
                    . This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for
+
<sup id="cite_ref-3" class="reference">
−
                     mechanism of the assembly
+
                            <a href="#cite_note-3">[8]
 +
                            </a>
 +
                     </sup>
  
−
                    <sup id="cite_ref-6" class="reference">
 
−
                        <a href="#cite_note-6">[6]
 
−
                        </a>
 
−
                    </sup>
 
−
                    .
 
−
                    <br>
 
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                    Here the scaffold protein(SP) is fused with a Strep-tagII for protein purification. The construct
 
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                    contains a pT7 promoter for the T7 polymerase, a lac operator, so expression can be induced with
 
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                    IPTG, and T7 Terminator.
 
  
−
                </p>
+
<sup id="cite_ref-4" class="reference">
 +
                            <a href="#cite_note-4">[9]
 +
                            </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.
  
−
                <h3> Methods</h3>
 
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                <h4>Cloning</h4>
 
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                <p>This constructed was cloned using PCR with overhang primers and <a
 
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                        href="https://2019.igem.org/Team:TU_Darmstadt/Project/Notebook" target="_blank">restriction and
 
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                        ligation</a> out of sfGFP-SP construct <a
 
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                        href="https://parts.igem.org/Part:BBa_K3187003">BBa_K3187003</a>.
 
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                    To verify the cloning,
 
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                    the sequence was controlled by sanger sequencing by Microsynth Seqlab.
 
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                </p>
 
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                <h4>Purification</h4>
 
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                <p>The SP was heterologously expressed in <i>E.&nbsp;coli</i> BL21 and purified with
 
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                    <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/Notebook" target="_blank">GE Healthcare
 
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                        ÄKTA Pure FPLC</a>.Strep-tag II was used as affinity tag.
 
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                </p>
 
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                <h4>SDS-Page and Western blot</h4>
 
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                <p>To verify that the Protein was produced, a SDS-Page <a
 
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                        href="https://2019.igem.org/Team:TU_Darmstadt/Project/Notebook" target="_blank">SDS-Page</a>
 
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                    followed by a
 
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                    <a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"
 
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                        target="_blank">Western blot</a> was performed.
 
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                </p>
 
  
 +
<sup id="cite_ref-5" class="reference">
 +
                            <a href="#cite_note-5">[10]
 +
                            </a>
 +
                    </sup>
  
 +
This fusion has to occur at the N&#8209;Terminus of the SP, because the C&#8209;Terminus is important for mechanism of the assembly.
  
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                <h4>Assembly</h4>
 
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                <p> The assembly was tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with
 
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                    ultracentrifugation <a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"
 
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                        target="_blank">ultracentrifugatione</a> and are visualized with
 
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                    <a href="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--Methoden.pdf"
 
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                        target="_blank">TEM</a>.
 
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                </p>
 
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                <h3>Results</h3>
 
  
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                <h4>Cloning and Expression</h4>
+
<sup id="cite_ref-6" class="reference">
−
                <p>The successful cloning was proven with sanger sequencing and production with a Western blot.
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                             <a href="#cite_note-6">[11]
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                </p>
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                            </a>  
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                <div style="text-align: center;">
+
                     </sup>  
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                    <img class="img-fluid center"
+
−
                        src="https://2019.igem.org/wiki/images/1/1a/T--TU_Darmstadt--westernplot_sp.jpeg"
+
−
                        style="max-width:60%" />
+
−
                    <div class="caption">
+
−
                        <p>
+
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                             <b>Figure 1:</b>
+
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                            Western blot of all produced and purified proteins.
+
−
                        </p>
+
−
                    </div>
+
−
                </div>
+
−
                <p>Fig. 1 shows that SP has a molecular weight of approximatley 30&nbsp;kDa. This is more than
+
−
                    the theoretical weight. Because the fusion prtoein of SP and sfGFP <a
+
−
                        href="https://parts.igem.org/Part:BBa_K3187003">(BBa_K3187003)</a> 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.</p>
+
  
  
 +
                </p>
 +
             
 +
                <h3> Methods</h3>
 +
                <h4>Cloning</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>
  
 +
                <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  
−
 
+
                     <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">western blot</a> was performed.
−
 
+
−
                 <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
+
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                    contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of
+
−
                    visually
+
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                    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>.
+
 
                 </p>
 
                 </p>
−
                <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" />
 
−
                    <div class="caption">
 
−
                        <p>
 
−
                            <b>Figure 2:</b> TEM picture of assembled VLPs
 
  
−
                        </p>
+
                <h3>Results</h3>  
−
                    </div>
+
                 <h4>Cloning and Expression</h4>
−
                 </div>
+
                 <p>The successful cloning was proven with Sanger sequencing and production 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%" />
−
                 <h2>References</h2>
+
                         <div class="caption">
−
 
+
                          <p>
−
                <ol class="references">
+
                          <b>Figure 1:</b>
−
                    <li id="cite_note-1">
+
                                Western blot of all produced and purified proteins.
−
                         <span class="mw-cite-backlink">
+
                            </p>
−
                            <a href="#cite_ref-1">↑</a>
+
                         </div>
−
                         </span>
+
                         </div>
−
                         <span class="reference-text">
+
                <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 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.
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                            W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray
+
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.  
−
                            diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387.
+
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                            <a rel="nofollow" class="external autonumber"
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                                href="https://doi.org/10.1016/0022-2836(76)90278-3">[1] </a>
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<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">
 +
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                            <span class="reference-text">
 +
                            Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics,
 +
                            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>
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                            <span class="reference-text">
 +
                            Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP,
 +
                            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>
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                             W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and
+
                             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, Nat. Struct.
+
                             maturation transition of bacteriophage P22 seen at subnanometer resolutions,Nature Structural Biology, 2003, 10: 131-135     
−
                            Biol. 2003, 10, 131.
+
                            <a rel="nofollow" class="external autonumber" href="https://www.nature.com/articles/nsb891">[3] </a>
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                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1038/nsb891">[2] </a>
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                             King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis.
+
                             Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein,
−
                             Nature 251:112-119.
+
                             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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                                href="https://www.nature.com/articles/251112a0">[3] </a>
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                            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 
 +
                            <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/pdf/10.1021/nn300545z">[5] </a>
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                             S. Casjens and R. Hendrix, (1988) "Control mechanisms in dsDNA bacteriophage assembly", in
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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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                             The Bacteriophages, volume 1, ed. R. Calendar, Plenum Press, p. 15-91.
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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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                                href="https://link.springer.com/chapter/10.1007/978-1-4684-5424-6_2">[4] </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.
 +
                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1016/0022-2836(76)90278-3">[7] </a>
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                              King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. 
 +
                            <a rel="nofollow" class="external autonumber" href="https://www.nature.com/articles/251112a0">[8] </a>
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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.
 +
                            <a rel="nofollow" class="external autonumber" href="https://link.springer.com/chapter/10.1007/978-1-4684-5424-6_2">[9] </a>
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                            Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas,
 
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                            Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle
 
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                            ,ACS Chemical Biology 2014 9 (2), 359-365
 
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                            Bacteriophage P22 Scaffolding Protein's Functional Domains
 
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                            , J. Mol. Biol. 2005, 348, 831.
 
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 +
,ACS Chemical Biology 2014 9 (2), 359-365
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, J. Mol. Biol. 2005, 348, 831.
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Latest revision as of 18:15, 21 October 2019


P22 Bacteriophage Scaffolding Protein

Profile

Name Scaffold protein
Base pairs 782
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). [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. [7] [3]
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. [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, because 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 proven with Sanger sequencing and production 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 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.

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
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 807
    Illegal XhoI site found at 1338
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1196
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1384
    Illegal SapI.rc site found at 105