Difference between revisions of "Team:NUDT CHINA/Design"

 
(18 intermediate revisions by 4 users not shown)
Line 1: Line 1:
 
{{NUDT_CHINA/content}}
 
{{NUDT_CHINA/content}}
 
<html>
 
<html>
 +
<div class="col-md-12 column" style="width:100%;height:250px;background-size:contain;0px -100px; background-image:url(https://static.igem.org/mediawiki/2018/6/66/T--NUDT_CHINA--banner_project_design.jpg);background-repeat:no-repeat;">
 +
  <p style="font-size:36px;margin: 0 0%;padding: 5% 0 0 10%;color: white;">Designed Protein Degradation Method Based on</p>
 +
<p style="font-size:36px;margin: 0 0%;padding: 0 0 0 10%;color: white;">Trim21 And Nanobody&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;--&nbsp;Design</p>
 +
</div>
  
 
<div class="container">
 
<div class="container">
Line 6: Line 10:
 
<div class="container" style="margin-top: 280px">
 
<div class="container" style="margin-top: 280px">
 
<div class="row" style="margin-top: 25px">
 
<div class="row" style="margin-top: 25px">
−
                                 <div class="col-md-push-1 col-md-10">
+
 
−
      <h1 style="font-size: 40px;font-weight: 900">Our Work</h1>
+
                                 <div class="col-md-push-1 col-md-10" alt = "this is a title model">
−
                                      <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Our Work</h2>
+
      <h1 style="font-size: 40px">PR PREDATOR-An improved protein degradation method based on ectopic expression of TRIM21 and recombinant antibody</h1></br></br>
−
<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
                                     
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
 
</div>
 
</div>
−
<div class="col-md-12 column" alt = "main_content">
+
 
 +
                                <div class="col-md-push-1 col-md-10" alt = "this is a paragraph model">
 +
<p>Design:</br></br>
 +
1. The design of ectopic expression of recombinant antibody.</br></br>
 +
To overcome the difficulty of expressing the multi-chain natural antibody in cells, we choose to express the single-domain antibody in our design. Single-domain antibodies, exampled by nanobody which identified as heavy-chain antibodies found in camelids, and scFv, a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, were able to bind selectively to a specific antigen with reduced amino acid number. However, the single-domain antibody lacks the constant Fc region, which is vital for the recognition of Tim21. To utilize the convenience of expressing single-domain antibody and bridge the recognition of Tim21 to single-domain antibody, in our design, the single-domain antibody was fused with human IgG-Fc domain. </br></br>
 +
By scanning reported single-domain antibodies in  literatures, we built a data sheet with all available single-domain antibodies and their target proteins enclosed. This data sheet can provide a sound basis for standardization of these nanobodies for further usage. The plasmid expressing the PR PREDATOR system was constructed by putting the coding regions of HA-Trim21 and nanobody-IgG Fc under CMV promoter. The HA-Trim21 and nanobody-IgG Fc regions were separated by P2A sequence to achieve bicistronic expression (Figure 1).  </br></br>
 +
Theoretically, when the recombinant plasmids were introduced into cells, the single-domain antibody will bind to target proteins with high affinity, forming tight complexes, and then Trim21 would bind to the Fc domain of antibodies with high affinity and recruits the ubiquitin-proteasome system to antibody-bound complex, finally leading to their destruction.
 +
 
 +
                                        </p>
 +
</div>     
 +
 
 +
 
 +
<div class="col-md-12 column" alt = "this is a picture model">
 
<div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
 
<div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/90/T--NUDT_CHINA--design1.jpg" />
−
<p alt="photo_detail" style="text-align: center;">This is the sign of the famous game!</p>
+
<p class="photo_detail">Figure 1. Schematic representation of the degradation process of our scheme</p>
 
</div>
 
</div>
−
+
<div class="col-md-push-1 col-md-10" alt = "this is a paragraph model">
−
<div class="col-md-6 col-md-push-3">
+
<p>2. Proof of concept<Br/><Br/>
−
<!-- <p class="text-center text-info">this is the sign of igem</p> -->
+
As a Proof of concept of our idea, we constructed GFP nanobody sequence into our PR PREDATOR system to make the GFP PREDATOR. A high-yield exogenous expression of GFP could provide us a visible approach to evaluate if our PREDATOR would work. For such matter, GFP expression plasmid and GFP PREDATOR plasmid were co-transfected in HEK293T or Hela cells, GFP fluorescence was observed under fluorescence microscope and GFP protein expression was detected by western blotting (Figure2A).
−
<p alt="photo_detail" style="text-align: center;"><em>This is the sign of the famous game!</em></p>
+
</p>
−
</div>
+
−
</div>
+
−
<div class="col-md-push-1 col-md-10">
+
−
<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
−
<blockquote style="border-left-color: #18d26e;">
+
−
Maybe you want to write <strong>something interesting thing</strong> here!
+
−
<small>Someone famous in <cite>Source Title</cite></small>
+
−
</blockquote>
+
−
<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
−
</div>
+
−
<div class="col-md-12 column" alt = "main_content">
+
−
<div class="col-md-6 col-md-push-3" alt="content_pictute">
+
−
<div class="row center-block">
+
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
−
<p alt="photo_detail" style="text-align: center;">This is the sign of the famous game!</p>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
<div class="col-md-push-1 col-md-10">
+
−
<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
−
<table class="table table-striped">
+
−
  <caption>The example table for show some data</caption>
+
−
  <thead>
+
−
<tr>
+
−
  <th>name</th>
+
−
  <th>city</th>
+
−
  <th>mail</th>
+
−
</tr>
+
−
  </thead>
+
−
  <tbody>
+
−
<tr>
+
−
  <td>Tanmay</td>
+
−
  <td>Bangalore</td>
+
−
  <td>560001</td>
+
−
</tr>
+
−
<tr>
+
−
  <td>Sachin</td>
+
−
  <td>Mumbai</td>
+
−
  <td>400003</td>
+
−
</tr>
+
−
<tr>
+
−
  <td>Uma</td>
+
−
  <td>Pune</td>
+
−
  <td>411027</td>
+
−
</tr>
+
−
  </tbody>
+
−
</table>
+
−
<p alt="content_add" style="font-size: 18px;">
+
−
Example block-level help text here.There are several variants of   derived from different organisms. In our project, we . The structure of this protein has been determined with and without  The  a lobe. The two lobes are positively charged towards the protein core to accommodate the negatively charged RNA. Each of these two lobes contains an RNase domain. At the  main is responsible for target cleavage. In contrast to other  two RNase domains of the NUC lobe are located at the outside of the protein, which is likely the reason collateral cleavage upon activation by binding to a matching target. These two domains have been labeled as red spots in Figure 2 and can be found at the interface between the green and pink domain.
+
−
</p>
+
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
  
−
<div class="col-md-push-1 col-md-4 thumbnail">
+
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/2/21/T--NUDT_CHINA--design1.2.jpg" />
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
<p class="photo_detail">Figure 2. Proof of concept and demonstration design of our project</p>
−
</div>
+
<div/>
−
<div class="col-md-push-3 col-md-4 thumbnail">
+
<div class="col-md-push-1 col-md-10" alt = "this is a paragraph model">
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
<p>3. Further demonstration<Br/><Br/>
−
</div>
+
For a more complicated demonstration, the endogenous receptor tyrosine-protein kinase erbB-3 was selected as our target protein which has been reported to play important roles in the poliferation, metastasis, and drug resistance in multiple cancers. Due to its significance, many antibodies of erbB3 have been used in clinical trials for the treatment of multiple kinds of cancer (such as MM-121, AMG 888 (U3-1287), TK-A3, TK-A4, AV-203, LJM716, MEHD7945A, MEHD7945A, MM-111 and MM-141). In our demonstration, the erbB-3 scFv antibody sequence would be expressed along with trim21 in MCF7 cells and directly functions on erbB-3 to bring down its level and inhibit MCF7 cells proliferation. The erbB3 protein expression could be detected by western blotting and influence on the proliferation of MCF7 cells could be verified by CCK-8 assay or colony formation assay (Figure2B).
−
</div>
+
<p/>
−
<div class="col-md-push-1 col-md-10">
+
        <div/>
−
<p alt="content_add" style="font-size: 18px;">
+
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
−
</p>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
+
−
<header class="section-header">
+
−
<h3>More details</h3>
+
−
</header>
+
−
<div class="col-md-12 column" alt="problem_answer">
+
−
<div class="panel-group" id="panel-1">
+
−
<div class="panel panel-success">
+
−
<div class="panel-heading text-center">
+
−
<a class="panel-title collapsed " data-toggle="collapse" data-parent="#panel-1" href="#panel-element-1"  style="color: #079209; font-size: 20px;text-decoration:none;">Question 1: What does the structure of domains look like?</a>
+
−
</div>
+
−
<div id="panel-element-1" class="panel-collapse collapse">
+
−
<div class="panel-body" style="font-size:16px;">
+
−
There are several variants of  derived from different organisms. In our project, we  . The structure of this protein has been determined with and without  The  a  lobe. The two lobes are positively charged towards the protein core to accommodate the negatively charged RNA. Each of these two lobes contains an RNase domain. At the  main is responsible for target cleavage. In contrast to other  two RNase domains of the NUC lobe are located at the outside of the protein, which is likely the reason collateral cleavage upon activation by binding to a matching target. These two domains have been labeled as red spots in Figure 2 and can be found at the interface between the green and pink domain.
+
−
</div>
+
−
</div>
+
−
</div>
+
−
<div class="panel panel-success">
+
−
<div class="panel-heading text-center">
+
−
<a class="panel-title collapsed" data-toggle="collapse" data-parent="#panel-1" href="#panel-element-2" style="color: #079209; font-size: 20px;text-decoration:none;">Collapsible Group Item #2</a>
+
−
</div>
+
−
<div id="panel-element-2" class="panel-collapse collapse">
+
−
<div class="panel-body" style="font-size:16px;">
+
−
Anim pariatur cliche...
+
−
</div>
+
−
</div>
+
−
</div>
+
 
</div>
 
</div>
 
</div>
 
</div>

Latest revision as of 03:53, 18 October 2018

Designed Protein Degradation Method Based on

Trim21 And Nanobody                 -- Design

PR PREDATOR-An improved protein degradation method based on ectopic expression of TRIM21 and recombinant antibody



Design:

1. The design of ectopic expression of recombinant antibody.

To overcome the difficulty of expressing the multi-chain natural antibody in cells, we choose to express the single-domain antibody in our design. Single-domain antibodies, exampled by nanobody which identified as heavy-chain antibodies found in camelids, and scFv, a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, were able to bind selectively to a specific antigen with reduced amino acid number. However, the single-domain antibody lacks the constant Fc region, which is vital for the recognition of Tim21. To utilize the convenience of expressing single-domain antibody and bridge the recognition of Tim21 to single-domain antibody, in our design, the single-domain antibody was fused with human IgG-Fc domain.

By scanning reported single-domain antibodies in literatures, we built a data sheet with all available single-domain antibodies and their target proteins enclosed. This data sheet can provide a sound basis for standardization of these nanobodies for further usage. The plasmid expressing the PR PREDATOR system was constructed by putting the coding regions of HA-Trim21 and nanobody-IgG Fc under CMV promoter. The HA-Trim21 and nanobody-IgG Fc regions were separated by P2A sequence to achieve bicistronic expression (Figure 1).

Theoretically, when the recombinant plasmids were introduced into cells, the single-domain antibody will bind to target proteins with high affinity, forming tight complexes, and then Trim21 would bind to the Fc domain of antibodies with high affinity and recruits the ubiquitin-proteasome system to antibody-bound complex, finally leading to their destruction.

300x200

Figure 1. Schematic representation of the degradation process of our scheme

2. Proof of concept

As a Proof of concept of our idea, we constructed GFP nanobody sequence into our PR PREDATOR system to make the GFP PREDATOR. A high-yield exogenous expression of GFP could provide us a visible approach to evaluate if our PREDATOR would work. For such matter, GFP expression plasmid and GFP PREDATOR plasmid were co-transfected in HEK293T or Hela cells, GFP fluorescence was observed under fluorescence microscope and GFP protein expression was detected by western blotting (Figure2A).

300x200

Figure 2. Proof of concept and demonstration design of our project

3. Further demonstration

For a more complicated demonstration, the endogenous receptor tyrosine-protein kinase erbB-3 was selected as our target protein which has been reported to play important roles in the poliferation, metastasis, and drug resistance in multiple cancers. Due to its significance, many antibodies of erbB3 have been used in clinical trials for the treatment of multiple kinds of cancer (such as MM-121, AMG 888 (U3-1287), TK-A3, TK-A4, AV-203, LJM716, MEHD7945A, MEHD7945A, MM-111 and MM-141). In our demonstration, the erbB-3 scFv antibody sequence would be expressed along with trim21 in MCF7 cells and directly functions on erbB-3 to bring down its level and inhibit MCF7 cells proliferation. The erbB3 protein expression could be detected by western blotting and influence on the proliferation of MCF7 cells could be verified by CCK-8 assay or colony formation assay (Figure2B).