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

 
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<p>Design:</br></br>
 
<p>Design:</br></br>
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By combining TRIM-AWAY strategy and ectopic expression of nanobody, we developed an improved method for degrading endogenous proteins in mammalian cells, named as PR PREDATOR.</br></br>
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1. The design of ectopic expression of recombinant antibody.</br></br>
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Two major barriers occur when attempting to selectively degrade endogenous proteins, one of which is the specificity, and the second is a powerful degradation mechanism to rely on. To address the specificity concern, we managed to use existing nanobody technique to achieve highly specific target recognition. By scanning existing patent files and literature reports, we built a data sheet with all available nanobodies and their target proteins enclosed. This data sheet can provide a sound basis for standardization of these nanobodies for further usage. The nanobodies are then fused with human IgG-Fc domain to bind Trim21, a powerful E3 ubiquitin-protein ligase we chose to mediate the degradation of target protein through proteasome. 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 1a).</br></br>
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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>
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We introduced the Recombinant plasmids into cells, nanobody specific binds to proteins with high affinity, forming tight complexes. Trim21 binds with high affinity to the Fc domain of antibodies and TRIM21 recruits the ubiquitin-proteasome system to antibody-bound complex, leading to their destruction. </br></br>
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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>
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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.  
  
 
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/7/75/T--NUDT_CHINA--design11111.png" />
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/90/T--NUDT_CHINA--design1.jpg" />
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<p class="photo_detail">Figure1. Schematic representation of the degration process of our scheme</p>
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<p class="photo_detail">Figure 1. Schematic representation of the degradation process of our scheme</p>
 
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<p>2. Proof of concept<Br/><Br/>
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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).
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<p class="photo_detail">Figure 2. Proof of concept and demonstration design of our project</p>
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
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<p class="photo_detail">Figure2.Workflow for our PR PREDATOR</p>
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<p>3. Further demonstration<Br/><Br/>
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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).
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As a Proof of concept of our idea, we constructed GFP nanobody sequence into our PR PREDATOR system.We choose GFP because the results of experiment is visible.</br></br>
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For a more complicated demonstration, the receptor tyrosine-protein kinase erbB-3 is selected. This protein is able to bind and form the heterodimerization with proteins from the erbB family, which is highly involved in proliferation and metastasis of cancer cells, including breast cancer, pancreatic adenocarcinoma and so on. It’s also implicated in chemotherapeutic resistance to NSCLC (non-small cell lung cancer) and another cancers through multiple signaling pathways, such as PI-3K/AKT and Jak/Stat signaling pathways. It has been proved that the downregulation of erbB-3 slows down the process several cancers. The most important reason of selecting erbB3 as our demonstration, is that many antibodies of erbB3 have been used in clinical trials for therapy in many kinds of cancer patients. 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 containment and inhibit MCF7 cells proliferation.</p>
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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).