Difference between revisions of "Part:BBa K3187000"
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| − | + | <div class="row"> | |
| − | + | <div class="col mx-2"> | |
| − | + | <h1>Profile</h1> | |
| − | + | <table style="width:80%"> | |
<tr> | <tr> | ||
| − | + | <td><b>Name</b></td> | |
| − | + | <td>Coat protein with LPETGG in pET24</td> | |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | + | <td><b>Base pairs</b></td> | |
| − | + | <td>1359</td> | |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | + | <td><b>Molecular weight</b></td> | |
| − | + | <td>49.0 kDa</td> | |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | + | <td><b>Origin</b></td> | |
| − | + | <td>Synthetic</td> | |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | + | <td><b>Parts</b></td> | |
| − | + | <td>Coat protein, LPETGG, T7 promoter, <i>lac</i>-operator, RBS, T7 terminator, Short Linker 5AA, | |
| + | Strep-tag II </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | + | <td><b>Properties</b></td> | |
| − | + | <td>Assembly with scaffold proteins to VLPs which can be modified exterior. </td> | |
</tr> | </tr> | ||
| − | + | </table> | |
| − | + | <h1>Sequence and Features</h1> | |
| + | </html> | ||
| − | + | <partinfo>BBa_K3187000 SequenceAndFeatures</partinfo> | |
| − | + | ||
| − | + | <html> | |
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| − | + | ||
| − | + | <h1> Usage and Biology</h1> | |
| − | + | ||
| − | + | <p>The P22 Virus-like particle (VLP) originates from the temperate bacteriophage P22. Its natural host is | |
| − | + | <i>Salmonella typhimurium</i>. | |
| − | + | 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 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 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.<sup id="cite_ref-3" class="reference"> | |
| − | + | <a href="#cite_note-3">[3]</a> | |
| − | + | </sup><br> | |
| − | + | 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.<sup | |
| − | + | id="cite_ref-4" class="reference"> | |
| − | + | <a href="#cite_note-4">[4]</a> | |
| − | + | </sup> | |
| − | + | When purified CPs and SPs are mixed, they self‑assemble into VLPs. </p> | |
| − | + | ||
| − | + | <p> 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.<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 = 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.<sup id="cite_ref-6" class="reference"> | |
| − | + | <a href="#cite_note-6">[6] </a> | |
| − | + | </sup> | |
| − | target="_blank" | + | </p> |
| − | + | ||
| − | <img | + | <p>The coat protein with LPETGG (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000" |
| − | + | target="_blank">BBa_K3187000)</a> | |
| − | + | consists of 452 amino acids, which are encoded by 1359 DNA base pairs. The | |
| − | + | whole | |
| − | + | protein has a mass of 49.0 kDa. Its relevant parts are the coat protein (CP) <a | |
| − | + | href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">(BBa_K3187017)</a> | |
| − | + | and the LPETGG sequence <a href="https://parts.igem.org/Part:BBa_K3187019" | |
| − | + | target="_blank">(BBa_K3187019)</a>. | |
| + | <br>LPETGG is a synthetic sequence that is recognized by the enzyme family Sortase A | ||
| + | and allows the coupling of CP with other peptides and proteins. For this, the sortase | ||
| + | cleaves between the amino acids threonine (T) and glycine (G), and threonine forms an amide | ||
| + | bond with another | ||
| + | polyG sequence. | ||
| + | <sup id="cite_ref-7" class="reference"> | ||
| + | <a href="#cite_note-7">[7] | ||
| + | </a> | ||
| + | </sup> | ||
| + | We used the Sortase A7M <a href="https://parts.igem.org/Part:BBa_K3187028" | ||
| + | target="_blank">(BBa_K3187028)</a> | ||
| + | and Sortase A5M <a href="https://parts.igem.org/Part:BBa_K3187016" | ||
| + | target="_blank">(BBa_K3187016)</a>. | ||
| + | The used polyG recognition sequence is composed of four glycines (GGGG) <a | ||
| + | href="https://parts.igem.org/Part:BBa_K3187018" target="_blank">(BBa_K3187018)</a> | ||
| + | |||
| + | |||
| + | <sup id="cite_ref-8" class="reference"> | ||
| + | <a href="#cite_note-8">[8] | ||
| + | </a> | ||
| + | </sup>. The assembled VLPs which consits of CP-LPETGG can be modified using sortase. | ||
| + | </p> | ||
| + | <a href="https://2019.igem.org/wiki/images/a/aa/T--TU_Darmstadt--modification_with_sfGFP.png" | ||
| + | target="_blank"> | ||
| + | <img src="https://2019.igem.org/wiki/images/a/aa/T--TU_Darmstadt--modification_with_sfGFP.png" | ||
| + | style="max-width:80%;" /> | ||
| + | </a> | ||
| + | |||
| + | <div class="caption"> | ||
<p> | <p> | ||
| − | + | <b> Figure 1:</b> | |
| − | + | Scheme of Sortase mediated P22-VLP modification. | |
</p> | </p> | ||
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</div> | </div> | ||
| + | </div> | ||
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| − | < | + | <p>Of course there are more parts necessary in order to express the CP‑LPETGG heterologously in |
| + | <i>E. coli</i> BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is | ||
| + | transcribed | ||
| + | into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the | ||
| + | protein. | ||
| + | The T7 promoter <a href="https://parts.igem.org/Part:BBa_K3187029" target="_blank">(BBa_K3187029)</a> | ||
| + | is recognized by the T7 polymerase. In order to regulate the protein production, the | ||
| + | <i>lac</i>‑operator <a href="https://parts.igem.org/Part:BBa_K3187029" | ||
| + | target="_blank">(BBa_K3187029)</a> was used. | ||
| + | Furthermore, a RBS <a href="https://parts.igem.org/Part:BBa_K3187029" target="_blank">(BBa_K3187029)</a> is | ||
| + | in the construct and | ||
| + | a Short Linker (5AA) <a href="https://parts.igem.org/Part:BBa_K3187030" | ||
| + | target="_blank">(BBa_K3187030)</a> | ||
| + | is found between CP and LPETGG. The T7 terminator <a href="https://parts.igem.org/Part:BBa_K3187032" | ||
| + | target="_blank">(BBa_K3187032)</a> and | ||
| + | Strep-tag II <a href="https://parts.igem.org/Part:BBa_K3187025" target="_blank">(BBa_K3187025)</a> are | ||
| + | located downstream of the coat protein CDS. | ||
| + | </p> | ||
| + | <h1> Methods</h1> | ||
| + | <h2>Cloning</h2> | ||
| + | <p>The CP-LPETGG was cloned into the pET24-backbone with <a | ||
| + | href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">restriction | ||
| + | and ligation</a> . | ||
| + | To do this, the CP‑LPETGG, as well as the T7 promoter and the | ||
| + | <i>lac</i>‑operator sequence, was ordered from Integrated DNA Technologies (IDT). To | ||
| + | verify the cloning, | ||
| + | the sequence was controlled by sanger sequencing by Microsynth Seqlab. | ||
| + | </p> | ||
| + | <h2>Purification</h2> | ||
| + | <p>The CP‑LPETGG was heterologously expressed in <i>E. coli</i> BL21 and purified with | ||
| + | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" | ||
| + | target="_blank">GE Healthcare ÄKTA Pure machine</a> | ||
| + | which is a machine for FPLC. The used affinity tag was Strep-tag II. | ||
| + | </p> | ||
| + | <h2>SDS-PAGE and western blot</h2> | ||
| + | <p>To verify that the CP-LPETGG was produced, a <a | ||
| + | href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SDS-PAGE</a> | ||
| + | followed by a | ||
| + | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" | ||
| + | target="_blank">western blot</a> was performed. | ||
| + | </p> | ||
| + | <h2>Sortase-mediated ligation</h2> | ||
| + | <p>In order to characterize CP‑LPETGG, different assays were performed. The possibility of modifying the | ||
| + | CP was tested with | ||
| + | mCherry and Sortase A7M. The Sortase A7M successfully linked mCherry and CP‑LPETGG. | ||
| + | The linkage was verified with a SDS‑PAGE. | ||
| + | To identify whether the Sortase A7M or Sortase A5M | ||
| + | <!-- WURDEN HIER BEIDE SORTASEN UNTERSUCHT?? --> | ||
| − | |||
| − | + | produce multimers of coat proteins with LPETGG‑tag, CP‑LPETGG and Sortase A7M and | |
| + | Sortase A5M | ||
| − | + | <!-- AUCH HIER: WURDEN BEIDE SORTASEN UNTERSUCHT? --> | |
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| − | + | were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS‑PAGE. | |
| − | + | </p> | |
| − | + | <h2>Assembly</h2> | |
| − | + | <p> The assembly was tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs were collected with | |
| + | <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" | ||
| + | target="_blank">ultracentrifugation</a> and | ||
| + | were visualized with transmission electron microscopy <a | ||
| + | href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">(TEM)</a>. | ||
| + | Therefore, the <i>in vivo</i> assembled VLPs are purified with size-exclusion chromatography | ||
| + | (<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SEC</a>) | ||
| + | (Sephadex-100 column) | ||
| + | <sup id="cite_ref-9" class="reference"> | ||
| + | <a href="#cite_note-9">[9] | ||
| + | </a> | ||
| + | </sup> | ||
| + | The diameter of VLPs was measured with dynamic light scattering (DLS) analysis. | ||
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| − | + | <h1>Results</h1> | |
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| − | + | <h2>Cloning and Expression</h2> | |
| + | <p>The successful cloning was confirmed with sanger sequencing. The purification was documented with an | ||
| + | chromatogram and the | ||
| + | successful production of the VLPs was confirmed with a western blot. | ||
| − | + | </p> | |
| − | + | <div> | |
| − | + | <a href="https://2019.igem.org/wiki/images/f/f0/T--TU_Darmstadt--Chrom_CP-LPETGG.png" target="_blank"> | |
| − | + | <img src="https://2019.igem.org/wiki/images/f/f0/T--TU_Darmstadt--Chrom_CP-LPETGG.png" | |
| + | style="max-width:40%" /> | ||
| + | </a> | ||
| + | |||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 2:</b> | ||
| + | Chromatogram of the purification of CP-LPETGG. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | <div> | ||
| + | <a href="https://2019.igem.org/wiki/images/0/03/T--TU_Darmstadt--Chrom_CP-LPETGG-Peak.png" | ||
| + | target="_blank"> | ||
| + | <img src="https://2019.igem.org/wiki/images/0/03/T--TU_Darmstadt--Chrom_CP-LPETGG-Peak.png" | ||
| + | style="max-width:40%" /> | ||
| + | </a> | ||
| + | |||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 3:</b> | ||
| + | Enlargement of the chromatogram for purification of CP-LPETGG. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <p>The chromatogram shows a peak for elution between 52 mL and 56 mL. The maximum | ||
| + | is found approximatley at 380 mAU (<b>Fig. 2 and 3</b>).</p> | ||
| + | |||
| + | |||
| + | <div> | ||
| + | <a href="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg" | ||
| + | target="_blank"> | ||
| + | <img class="img-fluid center" | ||
| + | src="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg" | ||
| + | style="max-width:40%" /> | ||
| + | </a> | ||
| + | |||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 4:</b> | ||
| + | Western blot of all produced and purified proteins. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <p><b>Fig. 4</b> shows western blot of produced and purified proteins. CP‑LPETGG was detected | ||
| + | using a Strep‑Tactin‑HRP conjugate. CP‑LPETGG can be seen at approximately 49 kDa. | ||
| + | Consequently, the successful production | ||
| + | was proven. </p> | ||
| + | |||
| + | |||
| + | |||
| + | <h2>Sortase-mediated ligation</h2> | ||
| + | |||
| + | |||
| + | <p> Want to know more about sortase-mediated ligation? Please have a look at our <a | ||
| + | href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase" target="_blank">wiki</a>.</p> | ||
| + | |||
| + | <p> | ||
| + | In order to test the funtion of the genetically fused sortase-tag LPETGG to the CP, we performed the linking reaction with CP-LPETGG and GGGG-mCherry. Resulting protein mix was applied to a | ||
| + | SDS-PAGE. | ||
| + | We saw products at the expected size (28 kDa + 49 kDa = 77 kDa) thus the requirement is | ||
| + | fulfilled. However, | ||
| + | a lot of additional bands appeared that we did not expect. These bands also appeared when only | ||
| + | Sortase A7M and CP were mixed. | ||
| + | </p> | ||
| + | |||
| + | <img class="img-fluid center" | ||
| + | src="https://2019.igem.org/wiki/images/1/1d/T--TU_Darmstadt--EnzymeSubstrate1.png" | ||
| + | style="max-width:50%" /> | ||
| + | </a> | ||
| + | |||
| + | |||
| + | <img class="img-fluid center" | ||
| + | src="https://2019.igem.org/wiki/images/7/76/T--TU_Darmstadt--Sortase7Mdiffprot.png" | ||
| + | style="max-width:50%" /> | ||
| + | </a> | ||
| + | |||
| + | |||
| + | <p> | ||
| + | <b> | ||
| + | Figure 5: </b> | ||
| + | <p><b>a)</b> Sortase A7M band is at expected height (17.85 kDa). | ||
| + | The two negative controls containing only GGGG-mCherry (28 kDa) | ||
| + | and CP-LPETGG (49 kDa) at the expected respective heights. <b>b)</b> Shown are sfGFP-SP and | ||
| + | CP-LPETGG each incubated with both Sortase A7M and Sortase A5M. | ||
| + | Both gels display multimers when coat and a sortase variant are in a sample together. | ||
| + | </p> | ||
| + | |||
| + | <p> | ||
| + | To investigate this issue, we had a look at the | ||
| + | literature and found a matching description in the publication of Patterson | ||
| + | et al.. They performed a similar experiment with P22 capsid proteins and observed | ||
| + | the same multimers in their SDS-PAGEs | ||
| + | </sup> | ||
| + | . Comparing both SDS-PAGEs, we came to the following assumption: | ||
| + | </p> | ||
| + | <p> | ||
| + | Because of the promiscuity of Sortase A7M to accept primary amines as substrates, as we | ||
| + | discussed previously, the formation of CP multimers occurs, unspecifically catalyzed by | ||
| + | Sortase A7M. | ||
| + | </p> | ||
| + | <p> | ||
| + | Parallel to these experiments, we successfully modified the exterior of pre-assembled VLPs <i>in | ||
| + | vitro</i> (<a href="https://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP" target="_blank">VLP | ||
| + | assembly</a>). These modified VLPs were homogenous and overall correctly assembled. | ||
| + | <b>Therefore, we conclude that the described multimer problem only occurs when Sortase A7M | ||
| + | encounters free CP.</b> | ||
| + | </p> | ||
<h2> Assembly</h2> | <h2> Assembly</h2> | ||
| − | <p>Ultracentrifugation was used to harvest VLPs after <i>in vivo</i> and <i>in vitro</i> assembly | + | <p>Ultracentrifugation was used to harvest VLPs consisting of CP (<a |
| + | href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">BBa_K3187017</a>) and SP after <i>in vivo</i> and <i>in vitro</i> assembly | ||
</p> | </p> | ||
<h3><i>In vivo assembled VLPs</i></h3> | <h3><i>In vivo assembled VLPs</i></h3> | ||
| − | <p>For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG, directly from cell broth we first | + | <p>For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG, directly from |
| − | lysed the cells by sonication and got rid of debris by two | + | cell broth we first |
| − | centrifugation steps at 12,000 x g. Afterwards ultracentrifugation with a sucrose cushion (35% w/v) at 150,000 x g was | + | lysed the cells by sonication and got rid of debris by two |
| − | used as a first concentration step. The resulting sediment contained fluorescent material which we suspected to contain | + | 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. | a concentrated fraction of VLPs. | ||
<div> | <div> | ||
| − | + | <a href="https://2019.igem.org/wiki/images/5/54/T--TU_DARMSTADT--UZ_invivo_2.png" | |
| − | + | target="_blank"> | |
| − | + | <img class="img-fluid" | |
| − | + | src="https://2019.igem.org/wiki/images/5/54/T--TU_DARMSTADT--UZ_invivo_2.png" | |
| − | + | style=max-width:40%;> | |
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</a> | </a> | ||
| + | <!--UZ-Pellet--> | ||
<div class="caption"> | <div class="caption"> | ||
| − | + | <p> | |
| − | + | <b> Figure 6:</b> | |
| − | + | Cell broth after ultracentrifugation. Supernatant containing sfGFP‑SP and CP while | |
| − | + | VLPs collected in the sediment.</p> | |
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | < | + | <p>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). | ||
| + | <sup id="cite_ref-12" class="reference"> | ||
| + | <a href="#cite_note-12">[12] | ||
| + | </a> | ||
| + | </sup> | ||
| + | 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 this | ||
| + | <sup id="cite_ref-12" class="reference"> | ||
| + | <a href="#cite_note-12">[12] | ||
| + | </a> | ||
| + | </sup> suggests. | ||
| − | + | <div> | |
| − | + | <a href="https://2019.igem.org/wiki/images/e/ee/T--TU_DARMSTADT--TEM_SEC.png" | |
| − | + | target="_blank"> | |
| − | + | <img class="img-fluid" | |
| − | + | src="https://2019.igem.org/wiki/images/e/ee/T--TU_DARMSTADT--TEM_SEC.png" | |
| − | + | style=max-width:40%;> | |
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</a> | </a> | ||
| + | <!--TEM Bild nach SEC--> | ||
<div class="caption"> | <div class="caption"> | ||
| − | + | <p> | |
| − | + | <b> Figure 7:</b> | |
| − | + | Intact P22-VLPs after size exclusion chromatography.</p> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
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| − | |||
| − | + | <h3><i>In vitro</i> assembled VLPs</h3> | |
| − | + | <p> The images of ultracentrifugation show 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, a sediment was clearly visible in the centrifuge tube, which we suspected to | |
| − | + | mainly contain VLPs. | |
| − | + | TEM was used to image capsids taken from the sediment. For increased | |
| − | <b>Fig. | + | 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 | |
| + | (<b>Fig. 8</b>). | ||
<div> | <div> | ||
| + | <a href="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png" | ||
| + | target="_blank"> | ||
| + | <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%" /> | ||
| + | </a> | ||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 8:</b> Ultracentrifugation of <i>in vitro</i>assembled VLPs. | ||
| + | |||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <p> The images taken via TEM show the assembled VLPs. VLPs only assemble with functional | ||
| + | coat proteins. Therefore, | ||
| + | the CPs produced using this part must be fully functional. The CPs assemble with | ||
| + | SPs and can be modified on the surface (<b>Fig. 8</b>). Moreover, CPs also assemble | ||
| + | without SPs | ||
| + | (<b>Fig. 9</b>). | ||
| + | </p> | ||
| + | <div> | ||
| + | <a href="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg" | ||
| + | target="_blank"> | ||
| + | <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%" /> | ||
| + | </a> | ||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 9:</b> | ||
| + | Assembly of only coat proteins with a LPETGG-tag. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <p><b>Fig. 9</b> shows that no scaffold proteins are necessary for assembly.</p> | ||
| + | |||
| + | <div> | ||
| + | <a href="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg" | ||
| + | target="_blank"> | ||
| + | <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%" /> | ||
| + | </a> | ||
| + | <div class="caption"> | ||
| + | <p> | ||
| + | <b>Figure 10:</b> | ||
| + | Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are | ||
| + | linked to sGFP. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <p><b>Fig. 10</b> shows that CP-LPETGG and SPs assemble to VLPs with structural integrity and that CP-LPETGG can | ||
| + | be modified for this process.</p> | ||
| + | <p>For more information about VLP assembly, please | ||
| + | visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP" | ||
| + | target="_blank">wiki</a>. </p> | ||
| + | |||
| + | <h2>Sortase-mediated ligation of GGGG-tagged proteins to the surface of the assembled P22 | ||
| + | VLP</h2> | ||
| + | <h3> Ultracentrifugation</h3> | ||
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<p> | <p> | ||
| − | + | We used ultracentrifugation over | |
| − | + | a sucrose cushion to separate freshly modified VLPs from monomeric capsid | |
| − | + | proteins, Sortase A5M, and sfGFP. After ultracentrifugation, a green | |
| + | fluorescent sediment was clearly visible (<b>Fig. 11</b>). This | ||
| + | is a strong indication that sortase has attached sfGFP to | ||
| + | the VLP exterior, as only assembled VLPs accumulate in the | ||
| + | sediment. | ||
| + | <sup id="cite_ref-8" class="reference"> | ||
| + | <a href="#cite_note-8">[8]</a></sup> We then prepared the ultracentrifugation | ||
| + | sediment for | ||
| + | transmission electron microscopy. Encouragingly, we observed numerous visually intact | ||
| + | VLPs. | ||
</p> | </p> | ||
| − | + | </div> | |
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| − | + | <div> | |
| − | + | <img class="img-fluid center" src="https://2019.igem.org/wiki/images/b/b4/T--TU_DARMSTADT--Mod_TEM2.png" | |
| − | + | style="max-width:60%" /> | |
| − | + | <div class="caption"> | |
| − | + | <p> | |
| − | + | <b>Figure 11:</b> | |
| − | + | Sediment containing P22-VLPs modified with sfGFP using | |
| − | + | SortaseA5M. Sediment was imaged in transmission electron | |
| − | + | microscope. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | </p> | |
| − | + | ||
| − | + | <h3>Dynamic light scattering</h3> | |
| − | + | ||
| − | + | <p>The hydrodynamic diameter of VLPs consisting of different protein combinations was measured with | |
| − | + | dynamic light scattering (DLS) analysis. | |
| − | + | In general hydrodynamic diameters depend on | |
| − | + | several properties like polarity and charges as well as size | |
| − | + | and shape. These properties can be summed up as the electrical properties of the system. | |
| − | + | <sup id="cite_ref-13" class="reference"> | |
| − | + | <a href="#cite_note-13">[13] </a> </sup>. | |
| − | + | </p> | |
| − | + | <div> | |
| − | + | <a href="https://2019.igem.org/wiki/images/9/90/T--TU_DARMSTADT--Hydro_radius.png" target="_blank"> | |
| − | + | <img class="img-fluid abstand" | |
| − | + | src="https://2019.igem.org/wiki/images/9/90/T--TU_DARMSTADT--Hydro_radius.png" style="max-width:30%;" /> | |
| − | + | </a> | |
| − | + | <!--Hydrodynamischer Radius--> | |
| − | + | <div class="caption"> | |
| − | + | <p> | |
| − | + | <b> Figure 12:</b> | |
| − | + | Influence of particle charge on hydrodynamic diameter. | |
| − | + | </p> | |
| − | + | </div> | |
| − | + | <div> | |
| − | + | <a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png" target="_blank"> | |
| − | + | <img class="img-fluid center" | |
| − | + | src="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png" | |
| − | + | style="max-width:50%" /> | |
| − | + | </a> | |
| − | + | <div class="caption"> | |
| − | + | <p> | |
| − | + | <b>Figure 13:</b> | |
| − | + | Diagram of DLS measurment of VLPs . | |
| − | + | </p> | |
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</div> | </div> | ||
</div> | </div> | ||
| + | <p> | ||
| + | We showed by dynamic | ||
| + | light scattering (DLS) analysis (<b>Fig. 13</b>) that capsids containing only CP are smaller than | ||
| + | P22-VLPs containing both CP and SP. This was | ||
| + | also confirmed by measuring VLPs and CP-only capsids in TEM images using ImageJ. Capsids which are only | ||
| + | composed of CP measured | ||
| + | average diameter of 53 nm±4.3 nm are significantly smaller than VLPs out of SP and CP measured | ||
| + | average diameter of 57 nm±3 nm | ||
| + | (n=20; p < 0.005). What also became clear is that the presence of the LPETGG tag does not affect | ||
| + | the size of the assembled CP-only capsid. </p> <p> | ||
| + | When we started to compare <b>sfGFP-modified VLPs</b> with | ||
| + | <b>non-modified</b> VLPs using <b>dynamic light scattering</b> (DLS), we | ||
| + | expected a difference in hydrodynamic radii because surface | ||
| + | modifications should further increase the hydration of the | ||
| + | particles as shown in | ||
| + | <b>Fig. 14</b><sup id="cite_ref-14" class="reference"> | ||
| + | <a href="#cite_note-14">[14] </a> | ||
| + | </sup>. | ||
| + | <div> | ||
| + | |||
| + | <a href="https://2019.igem.org/wiki/images/5/58/T--TU_DARMSTADT--DLS_VLPs_vs_VLPs.png" | ||
| + | target="_blank"> | ||
| + | <img class="img-fluid center" | ||
| + | src="https://2019.igem.org/wiki/images/5/58/T--TU_DARMSTADT--DLS_VLPs_vs_VLPs.png" | ||
| + | style="max-width:50%;" /> | ||
| + | </a> | ||
| + | <!--DLS mit allen--> | ||
| + | |||
| + | <p> | ||
| + | <b> Figure 14:</b> | ||
| + | Dynamic light scattering analysis. Hydrodynamic diameters of | ||
| + | different P22-VLP species. | ||
| + | </p> | ||
| + | |||
| + | |||
| + | <p> As described | ||
| + | <a href="https://2019.igem.org/Team:TU_Darmstadt/Safety" target="_blank">here</a>, non-modified | ||
| + | VLPs showed hydrodynamic diameters of | ||
| + | approximately 112.4 nm ± 41.3 nm. In comparison, | ||
| + | modified capsids showed an average hydrodynamic diameter of | ||
| + | 1446 nm. In our case, the drastically elevated hydrodynamic diameter of the | ||
| + | P22-VLP linked to sfGFP may result from strong hydration since | ||
| + | wild type sfGFP is multiply negatively charged | ||
| + | <sup id="cite_ref-15" class="reference"> | ||
| + | <a href="#cite_note-15">[15] </a> | ||
| + | </sup>. This probably leads to a tremendous charge density all over the surface. | ||
| + | Another possible reason could be the formation of sfGFP dimers | ||
| + | attached to the VLPs. | ||
| + | </p> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <p> | ||
| + | In order to demonstrate the integrity of our modified VLPs we | ||
| + | used capsids from the same sample for DLS and electron | ||
| + | microscopy which confirms the presence of intact VLPs. The | ||
| + | size distribution shows that they still pose a monodisperse | ||
| + | species, even though their hydrodynamic diameter is increased compared to | ||
| + | unmodified VLPs or capsids containing only CP. | ||
| + | </p> | ||
| + | |||
| + | <p>For more information about VLP assembly, please | ||
| + | visit our <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/VLP_Modification" | ||
| + | target="_blank">wiki</a>. </p> | ||
| + | |||
| + | <h2>References</h2> | ||
| + | <ol class="references"> | ||
| + | <li id="cite_note-1"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-1">↑</a> | ||
| + | </span> | ||
| + | <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> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-2"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-2">↑</a> | ||
| + | </span> | ||
| + | <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> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-3"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <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> | ||
| + | <li id="cite_note-4"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-4">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional | ||
| + | domains of bacteriophage P22 | ||
| + | scaffolding protein, | ||
| + | Journal of Molecular Biology, 1998, Volume 281: 69‑79 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://www.sciencedirect.com/science/article/pii/S0022283698919179">[4] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-5"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-5">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | 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" target="_blank">[5] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-6"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-6">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | 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 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299408/" target="_blank">[6] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-7"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-7">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, | ||
| + | Floris J. Bikker, Willem J. B. van Wamel, | ||
| + | Enno C. I. Veerman, | ||
| + | Synthetic LPETG-Containing Peptide Incorporation in the <i>Staphylococcus aureus</i> | ||
| + | Cell-Wall in a Sortase A- and Growth | ||
| + | Phase-Dependent Manner, plos one, 19.02.2014 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://doi.org/10.1371/journal.pone.0089260" target="_blank">[7] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | |||
| + | <li id="cite_note-8"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-8">↑</a> | ||
| + | </span> | ||
| + | <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#!divAbstractcite_note-1" | ||
| + | target="_blank">[8] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-9"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-9">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme | ||
| + | encapsulation | ||
| + | inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://pubs.acs.org/doi/abs/10.1021/nn300545z" target="_blank">[9] | ||
| + | </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-10"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-10">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, | ||
| + | Rosengren KJ, Mulvenna JP, Schroeder CI, | ||
| + | Craik DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A, | ||
| + | Journal of biological chemistry, 2014, 289, 627-6638 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="http://www.jbc.org/content/289/10/6627.long " target="_blank">[10] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-11"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-11">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, | ||
| + | Chenggang Wu, Neda Jooya, | ||
| + | Andrzej Joachimiak, Asis Das, Hung Ton-That‡2, A Disulfide Bond-forming | ||
| + | Machine | ||
| + | Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium | ||
| + | Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="http://www.jbc.org/content/290/35/21393.long" target="_blank">[11] </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-12"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-12">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme | ||
| + | encapsulation | ||
| + | inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://pubs.acs.org/doi/abs/10.1021/nn300545z">[12] | ||
| + | </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-13"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-13">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and | ||
| + | M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like | ||
| + | Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2): | ||
| + | 155 | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409934/" target="_blank">[13] | ||
| + | </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-14"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-14">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | https://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ | ||
| + | pharmaceuticals/ viruses-virus-like-particles/ | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://www.horiba.com/uk/scientific/products/particle-characterization/applications/pharmaceuticals/viruses-virus-like-particles/" | ||
| + | target="_blank">[14] | ||
| + | </a> | ||
| + | </span> | ||
| + | </li> | ||
| + | <li id="cite_note-15"> | ||
| + | <span class="mw-cite-backlink"> | ||
| + | <a href="#cite_ref-15">↑</a> | ||
| + | </span> | ||
| + | <span class="reference-text"> | ||
| + | Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E., | ||
| + | DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge | ||
| + | shielding prevents aggregation of supercharged GFP variants at | ||
| + | high protein concentration. Molecular pharmaceutics, 14(10), | ||
| + | 3269-3280. | ||
| + | <a rel="nofollow" class="external autonumber" | ||
| + | href="https://www.ncbi.nlm.nih.gov/pubmed/28870080" target="_blank">[15] | ||
| + | </a> | ||
| + | </span> | ||
| + | |||
| + | </ol> | ||
| + | |||
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| + | </div> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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Latest revision as of 23:00, 21 October 2019
P22 Bacteriophage Coat Protein with LPETGG Tag for Sortase-mediated Ligation
Profile
| Name | Coat protein with LPETGG in pET24 |
| Base pairs | 1359 |
| Molecular weight | 49.0 kDa |
| Origin | Synthetic |
| Parts | Coat protein, LPETGG, T7 promoter, lac-operator, RBS, T7 terminator, Short Linker 5AA, Strep-tag II |
| Properties | Assembly with scaffold proteins to VLPs which can be modified exterior. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1491
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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 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 coat protein with LPETGG (CP-LPETGG BBa_K3187000)
consists of 452 amino acids, which are encoded by 1359 DNA base pairs. The
whole
protein has a mass of 49.0 kDa. Its relevant parts are the coat protein (CP) (BBa_K3187017)
and the LPETGG sequence (BBa_K3187019).
LPETGG is a synthetic sequence that is recognized by the enzyme family Sortase A
and allows the coupling of CP with other peptides and proteins. For this, the sortase
cleaves between the amino acids threonine (T) and glycine (G), and threonine forms an amide
bond with another
polyG sequence.
[7]
We used the Sortase A7M (BBa_K3187028)
and Sortase A5M (BBa_K3187016).
The used polyG recognition sequence is composed of four glycines (GGGG) (BBa_K3187018)
[8]
. The assembled VLPs which consits of CP-LPETGG can be modified using sortase.
Figure 1: Scheme of Sortase mediated P22-VLP modification.
Of course there are more parts necessary in order to express the CP‑LPETGG heterologously in E. coli BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is transcribed into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the protein. The T7 promoter (BBa_K3187029) is recognized by the T7 polymerase. In order to regulate the protein production, the lac‑operator (BBa_K3187029) was used. Furthermore, a RBS (BBa_K3187029) is in the construct and a Short Linker (5AA) (BBa_K3187030) is found between CP and LPETGG. The T7 terminator (BBa_K3187032) and Strep-tag II (BBa_K3187025) are located downstream of the coat protein CDS.
Methods
Cloning
The CP-LPETGG was cloned into the pET24-backbone with restriction and ligation . To do this, the CP‑LPETGG, as well as the T7 promoter and the lac‑operator sequence, was ordered from Integrated DNA Technologies (IDT). To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.
Purification
The CP‑LPETGG was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for 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.
Sortase-mediated ligation
In order to characterize CP‑LPETGG, different assays were performed. The possibility of modifying the CP was tested with mCherry and Sortase A7M. The Sortase A7M successfully linked mCherry and CP‑LPETGG. The linkage was verified with a SDS‑PAGE. To identify whether the Sortase A7M or Sortase A5M produce multimers of coat proteins with LPETGG‑tag, CP‑LPETGG and Sortase A7M and Sortase A5M were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS‑PAGE.
Assembly
The assembly was tested in vivo and in vitro. The assembled VLPs were collected with ultracentrifugation and were visualized with transmission electron microscopy (TEM). Therefore, the in vivo assembled VLPs are purified with size-exclusion chromatography (SEC) (Sephadex-100 column) [9] The diameter of VLPs was measured with dynamic light scattering (DLS) analysis.
Results
Cloning and Expression
The successful cloning was confirmed with sanger sequencing. The purification was documented with an chromatogram and the successful production of the VLPs was confirmed with a western blot.
Figure 2: Chromatogram of the purification of CP-LPETGG.
Figure 3: Enlargement of the chromatogram for purification of CP-LPETGG.
The chromatogram shows a peak for elution between 52 mL and 56 mL. The maximum is found approximatley at 380 mAU (Fig. 2 and 3).
Figure 4: Western blot of all produced and purified proteins.
Fig. 4 shows western blot of produced and purified proteins. CP‑LPETGG was detected using a Strep‑Tactin‑HRP conjugate. CP‑LPETGG can be seen at approximately 49 kDa. Consequently, the successful production was proven.
Sortase-mediated ligation
Want to know more about sortase-mediated ligation? Please have a look at our wiki.
In order to test the funtion of the genetically fused sortase-tag LPETGG to the CP, we performed the linking reaction with CP-LPETGG and GGGG-mCherry. Resulting protein mix was applied to a SDS-PAGE. We saw products at the expected size (28 kDa + 49 kDa = 77 kDa) thus the requirement is fulfilled. However, a lot of additional bands appeared that we did not expect. These bands also appeared when only Sortase A7M and CP were mixed.
Figure 5:
a) Sortase A7M band is at expected height (17.85 kDa). The two negative controls containing only GGGG-mCherry (28 kDa) and CP-LPETGG (49 kDa) at the expected respective heights. b) Shown are sfGFP-SP and CP-LPETGG each incubated with both Sortase A7M and Sortase A5M. Both gels display multimers when coat and a sortase variant are in a sample together.
To investigate this issue, we had a look at the literature and found a matching description in the publication of Patterson et al.. They performed a similar experiment with P22 capsid proteins and observed the same multimers in their SDS-PAGEs . Comparing both SDS-PAGEs, we came to the following assumption:
Because of the promiscuity of Sortase A7M to accept primary amines as substrates, as we discussed previously, the formation of CP multimers occurs, unspecifically catalyzed by Sortase A7M.
Parallel to these experiments, we successfully modified the exterior of pre-assembled VLPs in vitro (VLP assembly). These modified VLPs were homogenous and overall correctly assembled. Therefore, we conclude that the described multimer problem only occurs when Sortase A7M encounters free CP.
Assembly
Ultracentrifugation was used to harvest VLPs consisting of CP (BBa_K3187017) and SP after in vivo and in vitro assembly
In vivo assembled VLPs
For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG, 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.
Figure 6: Cell broth after ultracentrifugation. Supernatant containing sfGFP‑SP and CP while VLPs collected in the sediment.
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 this [12] suggests.
Figure 7: Intact P22-VLPs after size exclusion chromatography.
In vitro assembled VLPs
The images of ultracentrifugation show 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, a sediment was clearly visible in the centrifuge tube, which we suspected to mainly contain VLPs. 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. 8).
Figure 8: Ultracentrifugation of in vitroassembled VLPs.
The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, the CPs produced using this part must be fully functional. The CPs assemble with SPs and can be modified on the surface (Fig. 8). Moreover, CPs also assemble without SPs (Fig. 9).
Figure 9: Assembly of only coat proteins with a LPETGG-tag.
Fig. 9 shows that no scaffold proteins are necessary for assembly.
Figure 10: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
Fig. 10 shows that CP-LPETGG and SPs assemble to VLPs with structural integrity and that CP-LPETGG can be modified for this process.
For more information about VLP assembly, please visit our wiki.
Sortase-mediated ligation of GGGG-tagged proteins to the surface of the assembled P22 VLP
Ultracentrifugation
We used ultracentrifugation over a sucrose cushion to separate freshly modified VLPs from monomeric capsid proteins, Sortase A5M, and sfGFP. After ultracentrifugation, a green fluorescent sediment was clearly visible (Fig. 11). This is a strong indication that sortase has attached sfGFP to the VLP exterior, as only assembled VLPs accumulate in the sediment. [8] We then prepared the ultracentrifugation sediment for transmission electron microscopy. Encouragingly, we observed numerous visually intact VLPs.
Figure 11: Sediment containing P22-VLPs modified with sfGFP using SortaseA5M. Sediment was imaged in transmission electron microscope.
Dynamic light scattering
The hydrodynamic diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis. In general hydrodynamic diameters depend on several properties like polarity and charges as well as size and shape. These properties can be summed up as the electrical properties of the system. [13] .
Figure 12: Influence of particle charge on hydrodynamic diameter.
Figure 13: Diagram of DLS measurment of VLPs .
We showed by dynamic light scattering (DLS) analysis (Fig. 13) that capsids containing only CP are smaller than P22-VLPs containing both CP and SP. This was also confirmed by measuring VLPs and CP-only capsids in TEM images using ImageJ. Capsids which are only composed of CP measured average diameter of 53 nm±4.3 nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57 nm±3 nm (n=20; p < 0.005). What also became clear is that the presence of the LPETGG tag does not affect the size of the assembled CP-only capsid.
When we started to compare sfGFP-modified VLPs with non-modified VLPs using dynamic light scattering (DLS), we expected a difference in hydrodynamic radii because surface modifications should further increase the hydration of the particles as shown in Fig. 14 [14] .
Figure 14: Dynamic light scattering analysis. Hydrodynamic diameters of different P22-VLP species.
As described here, non-modified VLPs showed hydrodynamic diameters of approximately 112.4 nm ± 41.3 nm. In comparison, modified capsids showed an average hydrodynamic diameter of 1446 nm. In our case, the drastically elevated hydrodynamic diameter of the P22-VLP linked to sfGFP may result from strong hydration since wild type sfGFP is multiply negatively charged [15] . This probably leads to a tremendous charge density all over the surface. Another possible reason could be the formation of sfGFP dimers attached to the VLPs.
In order to demonstrate the integrity of our modified VLPs we used capsids from the same sample for DLS and electron microscopy which confirms the presence of intact VLPs. The size distribution shows that they still pose a monodisperse species, even though their hydrodynamic diameter is increased compared to unmodified VLPs or capsids containing only CP.
For more information about VLP assembly, please visit our wiki.
References
- ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412‑10414 [2]
- ↑ Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nature Structural Biology, 2003, 10: 131‑135 [3]
- ↑ Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69‑79 [4]
- ↑ Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 [5]
- ↑ P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures., Biophysical Journal, 1998, 74: 559-568 [6]
- ↑ Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman, Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner, plos one, 19.02.2014 [7]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [8]
- ↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [9]
- ↑ Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, Craik DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A, Journal of biological chemistry, 2014, 289, 627-6638 [10]
- ↑ Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, Chenggang Wu, Neda Jooya, Andrzej Joachimiak, Asis Das, Hung Ton-That‡2, A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 [11]
- ↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [12]
- ↑ J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2): 155 [13]
- ↑ https://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ pharmaceuticals/ viruses-virus-like-particles/ [14]
- ↑ Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E., DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge shielding prevents aggregation of supercharged GFP variants at high protein concentration. Molecular pharmaceutics, 14(10), 3269-3280. [15]