Difference between revisions of "Part:BBa K3187000"
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</a> | </a> | ||
</sup> | </sup> | ||
− | We used the Sortase A7M <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">(BBa_K3187028)</a> | + | 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>. | + | 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>. | 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>. | ||
− | <br>The CP is originally found in the bacteriophage P22 and forms its capsid with the scaffold protein (SP) | + | <br>The CP is originally found in the bacteriophage P22 and forms its capsid with the scaffold protein (SP) |
<a href="https://parts.igem.org/Part:BBa_K3187021"target="_blank">(BBa_K3187021)</a>. | <a href="https://parts.igem.org/Part:BBa_K3187021"target="_blank">(BBa_K3187021)</a>. | ||
Heterologously expressed, coat proteins and scaffold poroteins assemble to a Virus-like particles (VLP). | Heterologously expressed, coat proteins and scaffold poroteins assemble to a Virus-like particles (VLP). | ||
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</p> | </p> | ||
<p>Of course there are more parts necessary in order to express the CP-LPETGG heterologously in | <p>Of course there are more parts necessary in order to express the CP-LPETGG heterologously in | ||
− | <i>E. coli</i> BL21. As a backbone, the pET24 | + | <i>E. coli</i> BL21. 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. | 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> | + | 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 | + | 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. | <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 | 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> | + | 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 | + | 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 | 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. | + | located downstream of the coat protein CDS. |
</p> | </p> | ||
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<h4>Cloning</h4> | <h4>Cloning</h4> | ||
<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> . | <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 | + | 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, | <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. | the sequence was controlled by sanger sequencing by Microsynth Seqlab. | ||
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<h4>Sortase-mediated Ligation</h4> | <h4>Sortase-mediated Ligation</h4> | ||
<p>In order to characterize CP-LPETGG, different assays were performed. The possibility of modifying the CP was tested with | <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 Sortase A7M. The Sortase A7M successfully linked mCherry and CP-LPETGG. |
− | The linkage was verified with a | + | The linkage was verified with a SDS-PAGE. |
− | To identify whether the | + | To identify whether the Sortase A7M or Sortase A5M |
− | were incubated for 3 h at 37 °C. | + | |
+ | <!-- 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? --> | ||
+ | |||
+ | |||
+ | were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS-PAGE. For more information, please have a look at our <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>. | ||
</p> | </p> | ||
<h4>Assembly</h4> | <h4>Assembly</h4> | ||
− | <p> The assembly was tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs | + | <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 | + | <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>. |
− | + | ||
The diameter of VLPs was measured with dynamic light scattering (DLS) analysis. | The diameter of VLPs was measured with dynamic light scattering (DLS) analysis. | ||
For more information look at our | For more information look at our | ||
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<h4>Cloning and Expression</h4> | <h4>Cloning and Expression</h4> | ||
− | <p>The successful cloning was | + | <p>The successful cloning was confirmed with sanger sequencing and successful production of the VLPs was confirmed with a western blot. |
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | <p><b>Fig. 1</b> shows that the band of the CP-LPETGG is | + | <p><b>Fig. 1</b> shows that the band of the CP-LPETGG is can be seen at approximately 49 kDa. Consequently, the successful production |
was proven. CP-LPETGG was detected with Strep-Tactin-HRP.</p> | was proven. CP-LPETGG was detected with Strep-Tactin-HRP.</p> | ||
Line 138: | Line 148: | ||
</p> | </p> | ||
<h4>Forming multimers</h4> | <h4>Forming multimers</h4> | ||
− | <p>The SDS-PAGE suggests that the CP-LPETGG does not form multimers, even if it is in high concentration. | + | <p>The SDS-PAGE |
+ | |||
+ | <!-- WELCHE SDS-PAGE? --> | ||
+ | |||
+ | |||
+ | |||
+ | suggests that the CP-LPETGG does not form multimers | ||
+ | |||
+ | |||
+ | <!-- WENN ES NUR CP-LPETGG IST? ALSO OHNE SORTASEN? --> | ||
+ | |||
+ | , even if it is in high concentration. | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
<a href="https://2019.igem.org/wiki/images/f/ff/T--TU_Darmstadt--Coat_%2B_BSA.png"target="_blank"> | <a href="https://2019.igem.org/wiki/images/f/ff/T--TU_Darmstadt--Coat_%2B_BSA.png"target="_blank"> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | <b>Fig. 2</b> shows the bands of BSA | + | <b>Fig. 2</b> shows the bands of BSA at approximately 66 kDa and the CP-LPETGG at approximately 49 kDa. When the proteins were combined, |
− | two bands, one of CP-LPETGG and one of BSA. Hence, no multimers were formed. | + | two bands can be seen, one of CP-LPETGG and one of BSA. Hence, no multimers were formed. |
</p> | </p> | ||
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</div> | </div> | ||
− | The SDS-PAGE shows multiple bands (<b>Fig. 3</b>), which relate to a higher molecular weight than mCherry or CP-LPETGG | + | The SDS-PAGE shows multiple bands (<b>Fig. 3</b>), which relate to a higher molecular weight than mCherry or CP-LPETGG themselves have. |
The bands located between 55 kDa and 70 kDa most likley show the linked CP-LPETGG and GGGG-mCherry, as we expected the product to be approximately | The bands located between 55 kDa and 70 kDa most likley show the linked CP-LPETGG and GGGG-mCherry, as we expected the product to be approximately | ||
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− | + | ||
− | Want to know more about how the modification works? Please have | + | Want to know more about how the modification works? Please have a look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>. |
</p> | </p> | ||
<p> | <p> | ||
Line 207: | Line 228: | ||
</a> | </a> | ||
</sup> | </sup> | ||
− | and the P22 Coat Protein accommodates a cysteine residue. | + | |
+ | |||
+ | <!-- FEHLT HIER EIN SATZTEIL, ODER GEHOERT DAS ZU OBEN? --> | ||
+ | |||
+ | |||
+ | and the P22 Coat Protein accommodates a cysteine residue. | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
<a href="https://static.igem.org/mediawiki/parts/c/c7/T--TU_Darmstadt--CP-LPETGG_Sortase.png"target="_blank"> | <a href="https://static.igem.org/mediawiki/parts/c/c7/T--TU_Darmstadt--CP-LPETGG_Sortase.png"target="_blank"> | ||
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<p> | <p> | ||
<b>Figure 2:</b> | <b>Figure 2:</b> | ||
− | SDS-PAGE of sortase-mediated linkage between several coat proteins with LPETGG tag. | + | SDS-PAGE of sortase-mediated linkage between several coat proteins with LPETGG-tag. |
</p> | </p> | ||
</div> | </div> | ||
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<h4> Assembly</h4> | <h4> Assembly</h4> | ||
− | <p> The images of ultracentrifugation | + | <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 | + | 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 | 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 | + | contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually |
− | intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly, | + | intact VLPs all over the sample, measuring a diameter of 60 nm or less (<b>Fig. 2</b>). For more information about VLP assembly, |
visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"target="_blank">wiki</a>. | visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"target="_blank">wiki</a>. | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
Line 241: | Line 267: | ||
</div> | </div> | ||
</p> | </p> | ||
− | <p> The images | + | <p> The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, |
− | the CPs produced using this part | + | the CPs produced using this part must be fully functional. The CPs assemble with |
− | SPs and | + | SPs and can be modified on the surface (<b>Fig. 4</b>). Moreover, CPs also assemble without SPs |
− | (Fig. 3). | + | (<b>Fig. = 3</b>). |
</p> | </p> | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
Line 253: | Line 279: | ||
<p> | <p> | ||
<b>Figure 3:</b> | <b>Figure 3:</b> | ||
− | Assembly of only coat proteins with a LPETGG tag. | + | Assembly of only coat proteins with a LPETGG-tag. |
</p> | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
− | <p>Fig. 3 shows that no scaffold proteins are necessary for assembly.</p> | + | <p><b>Fig. 3</b> shows that no scaffold proteins are necessary for assembly.</p> |
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
Line 271: | Line 297: | ||
</div> | </div> | ||
− | <p>Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and CP-LPETGG can be modified for this process.</p> | + | <p><b>Fig. 4</b> shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.</p> |
− | <p>The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering | + | <p>The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis. |
− | + | ||
<div style="text-align: center;"> | <div style="text-align: center;"> | ||
<a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png"target="_blank"> | <a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png"target="_blank"> | ||
Line 282: | Line 307: | ||
<p> | <p> | ||
<b>Figure 5:</b> | <b>Figure 5:</b> | ||
− | Diagram of DLS measurment of VLPs | + | Diagram of DLS measurment of VLPs . |
</p> | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
− | As shown in the diagram, VLPs which only consist of | + | As shown in the diagram, VLPs which only consist of coat proteins with LPETGG-tag and VLPs made out of coat proteins |
− | without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). VLPs with both proteins have a diameter | + | without a tag are smaller than P22-VLPs containing CP and SP (<b>Fig. 5</b>). VLPs with both proteins have a diameter |
− | about 112 nm. Consequently, the LPETGG tag does not disturb the assembly of coat proteins. | + | of about 112 nm. Consequently, the LPETGG-tag does not disturb the assembly of coat proteins. |
</p> | </p> | ||
Line 299: | Line 324: | ||
</span> | </span> | ||
<span class="reference-text"> | <span class="reference-text"> | ||
− | Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman, | + | 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 | + | 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 | + | Phase-Dependent Manner, plos one, 19.02.2014 |
<a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260">[1] </a> | <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260">[1] </a> | ||
</span> | </span> | ||
Line 311: | Line 336: | ||
</span> | </span> | ||
<span class="reference-text"> | <span class="reference-text"> | ||
− | Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration | + | Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration |
− | into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 | + | 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">[2] </a> | <a rel="nofollow" class="external autonumber" href="https://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc46517a#!divAbstractcite_note-1">[2] </a> | ||
</span> | </span> | ||
Line 321: | Line 346: | ||
</span> | </span> | ||
<span class="reference-text"> | <span class="reference-text"> | ||
− | Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, | + | 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, | + | 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 ">[3] </a> | <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/289/10/6627.long ">[3] </a> | ||
</span> | </span> | ||
Line 332: | Line 356: | ||
</span> | </span> | ||
<span class="reference-text"> | <span class="reference-text"> | ||
− | Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, Chenggang Wu, Neda Jooya, | + | 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 | + | 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 | Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium | ||
− | Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 | + | 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">[4] </a> | <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/290/35/21393.long">[4] </a> | ||
</span> | </span> |
Revision as of 10:22, 20 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-tagII |
Properties | Assembly with scaffold proteins to VLPs which can be modified exterior. |
Usage and Biology
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.
[1]
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).
The CP is originally found in the bacteriophage P22 and forms its capsid with the scaffold protein (SP)
(BBa_K3187021).
Heterologously expressed, coat proteins and scaffold poroteins assemble to a Virus-like particles (VLP).
[2]
Of course there are more parts necessary in order to express the CP-LPETGG heterologously in E. coli BL21. 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.
Forming multimers
To test whether coat proteins with a LPETGG tag form stable multimers, the concentration of CP-LPETGG was increased in the presence of the protein BSA. The concentrated protein solution was heated up to 95 °C and a SDS-PAGE was performed to verify the stability of multimers.
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. For more information, please have a look at our wiki.
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). The diameter of VLPs was measured with dynamic light scattering (DLS) analysis. For more information look at our wiki.
Results
Cloning and Expression
The successful cloning was confirmed with sanger sequencing and successful production of the VLPs was confirmed with a western blot.
Fig. 1 shows that the band of the CP-LPETGG is can be seen at approximately 49 kDa. Consequently, the successful production was proven. CP-LPETGG was detected with Strep-Tactin-HRP.
Forming multimers
The SDS-PAGE suggests that the CP-LPETGG does not form multimers , even if it is in high concentration.
Fig. 2 shows the bands of BSA at approximately 66 kDa and the CP-LPETGG at approximately 49 kDa. When the proteins were combined, two bands can be seen, one of CP-LPETGG and one of BSA. Hence, no multimers were formed.Sortase-medited Reaction
The possibility of modification was shown with a SDS-PAGE, which shows GGGG-mCherries linked to several CP-LPETGG.
The SDS-PAGE shows multiple bands (Fig. 3), which relate to a higher molecular weight than mCherry or CP-LPETGG themselves have. The bands located between 55 kDa and 70 kDa most likley show the linked CP-LPETGG and GGGG-mCherry, as we expected the product to be approximately XY kDa. Want to know more about how the modification works? Please have a look at our wiki.The results on the SDS-PAGE of testing sortase-mediated linkage between coat proteins with LPETGG-tag with Sortase A7M and Sortase A5M (Fig. 2) suggested that Sortase A7M and Sortase A5M produce CP-LPETGG multimers, because wild type Sortase A is able to link two proteins together via disulfide bridges. [3] [4] and the P22 Coat Protein accommodates a cysteine residue.
Assembly
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. 2). For more information about VLP assembly, visit our wiki.
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. 4). Moreover, CPs also assemble without SPs (Fig. = 3).
Fig. 3 shows that no scaffold proteins are necessary for assembly.
![](https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg)
Figure 4: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.
The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis.
As shown in the diagram, VLPs which only consist of coat proteins with LPETGG-tag and VLPs made out of coat proteins without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). VLPs with both proteins have a diameter of about 112 nm. Consequently, the LPETGG-tag does not disturb the assembly of coat proteins.References
- ↑ 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 [1]
- ↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [2]
- ↑ 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 [3]
- ↑ 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 [4]
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]