Difference between revisions of "Part:BBa K4768002"
 
(18 intermediate revisions by 2 users not shown)
Line 26: Line 26:
 
             style="width:90%;"
 
             style="width:90%;"
 
             src="https://static.igem.wiki/teams/4768/wiki/parts/bba-k4768002.png">  
 
             src="https://static.igem.wiki/teams/4768/wiki/parts/bba-k4768002.png">  
−
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 1: Anti-HER2 nanobody part</b></i></span></figcaption>
+
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 1: Anti-HER2 Nanobody Section. "VHH-HER2" corresponds to the sequence of the Anti-HER2 Nanobody.</b></i></span></figcaption>
 
         </figure>
 
         </figure>
 
</div>
 
</div>
−
<p>Toulouse-INSA-UPS 2023 designed this part in order to decorate the liposome for the anchoring to cancer cells. To express and purify these Nanobodies Anti-HER2 (nb Anti-HER2), we used the <i>E. coli</i> strain BL21(DE3). This strain was obtained from by Adilya Dagkesamanskaya, a researcher from the Toulouse Biotechnology Institute (TBI). The plasmide pET26b_pelB-HER2-tev-stops which contained our part were transformed into BL21(DE3) by Adilya Dagkesamanskaya research team.</p>
+
<p>Toulouse-INSA-UPS 2023 designed this part to functionalize the liposome with anti-HER2 nanobodies for anchoring to cancer cells. To express and purify this nanobody, hereafter called Anti-HER2-nb , we used the <i>E. coli</i> strain BL21(DE3) with the plasmid pET26b_pelB-HER2-tev-stops, provided to us by Adilya Dagkesamanskaya, a researcher at the Toulouse Biotechnology Institute (TBI). This plasmid contains the gene for Anti-HER2-nb fused to a signal peptide gene called pelB leader, along with a His-tag for purification. The signal peptide was fused to Anti-HER2-nb for periplasmic targeting, which promotes disulfide bond formation.</p>
  
−
<h2>Construction expression and purification</h2>
+
<h2>Protein expression and purification </h2>
−
<p>The Calipso part BBa_K4768002  contained a gene for nanobody Anti-HER2 fused to a signal peptide gene called pelB leader, as well as a Histag for purification. The signal peptide was fused to nb Anti-HER2 with the aim of achieving periplasmic expression. 
+
<p><I>E. coli</I> expression of Anti-HER2-nb was induced using IPTG, and purification was carried out using Cobalt resin (TALON® Metal Affinity Resin). Two separate batches of protein expression and purification were prepared, and both were successful. </p>
−
The expression of nb Anti-HER2 was induced using IPTG, and purification was carried out using Cobalt resin (TALON® Metal Affinity Resin). </p>
+
−
<p>Two separate batches of protein expression and purification were performed in duplicate, and both were successful.  
+
−
</p>
+
  
 
<div align="center">
 
<div align="center">
Line 41: Line 38:
 
             <img
 
             <img
 
             class="d-block"
 
             class="d-block"
−
             style="width:90%;"
+
             style="width:60%;"
 
             src="https://static.igem.wiki/teams/4768/wiki/module1/figure1-module1.png">  
 
             src="https://static.igem.wiki/teams/4768/wiki/module1/figure1-module1.png">  
−
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 2: SDS-page migration  (15% acrylamid) on purification of periplasmic extract and Coomassie Blue revelation. Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/ 500)
+
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 1: SDS-PAGE analysis (15% acrylamide) of protein samples after purification of periplasmic extract and Coomassie Blue staining.</b> Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/ 500).</i></span></figcaption>
−
</b></i></span></figcaption>
+
 
         </figure>
 
         </figure>
 
</div>
 
</div>
  
−
<p>The expected size of Anti-HER2 nb is approximately 17 kDa. In figure 2  we can observe a clear band around this size in the extract and flowthrough, demonstrating expression of the nanobody Anti-HER2. The presence of a similar band in the negative control (No IPTG, lane 2, gel 2 in Figure 1) probably reflects an uncontrolled expression of the nanobody due to promoter leakage. Approximately 2 mL of the nanobody at 17.65 µM was produced for each sample.</p>
+
<p>The expected size of Anti-HER2 nb is approximately 17 kDa. In Figure 1 one can observe a clear band around this size in the extract and flowthrough, demonstrating expression of the nanobody Anti-HER2. The presence of a similar band in the negative control (No IPTG, lane 2, gel 2 in Figure 1) probably reflects an uncontrolled expression of the nanobody due to promoter leakage. Approximately 2 mL of the nanobody at 17.65 µM was produced for each sample.</p>
  
  
 
<h2>Characterisation</h2>
 
<h2>Characterisation</h2>
−
<h3>Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells</h3>
+
<p>To test whether liposomes can anchor to cancer cells via Anti-HER2-nb, we prepared fluorescent liposomes with a diameter of 400 nm. These liposomes contain DGS lipids, which allowed us to attach Anti-HER2-nb (<a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">See the protocol here</a>). We used HER2-positive colorectal adenocarcinoma cells called Caco-2 cells to test nanobody-mediated liposome anchoring (<a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">See the protocol here</a>). Figure 3 shows a microscopy image of adherent and non-adherent Caco-2 cells observed in Brightfield. For educational purposes, we added labels to the image to highlight some features of living eukaryotic cells that can be seen with a regular optical microscope.</p>
−
<p>400 nm fluorescent liposomes were prepared and coated with anti-HER2 nb, <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. Colorectal adenocarcinoma cells Caco-2, which are HER2-positive, were used to test the anchoring of liposomes by Anti-HER2 nb <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. Figure 1 shows a microscopy image of <b>adherent and non-adherent Caco-2 cells</b> observed in brightfield. For education purposes we appended on the image some features of living eukaryotic cells that can be distinguished with a standard optical microscope.</p>
+
 
<div align="center">
 
<div align="center">
 
         <figure class="normal mx-auto">     
 
         <figure class="normal mx-auto">     
Line 60: Line 55:
 
             style="width:90%;"
 
             style="width:90%;"
 
             src="https://static.igem.wiki/teams/4768/wiki/module1/figure2-module1.png">  
 
             src="https://static.igem.wiki/teams/4768/wiki/module1/figure2-module1.png">  
−
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification. </b></i></span></figcaption>
+
             <figcaption class="normal"><span class="titre-image"><i><b>Figure 3:</b> Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification.  
 +
</i></span></figcaption>
 
         </figure>
 
         </figure>
 
</div>
 
</div>
−
<p>Fluorescent liposomes were incubated on top of Caco-2 cells as described in our <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">Protocol page</a>. Figure 3 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.</p>
+
<p>
 +
Fluorescent liposomes were incubated on top of Caco-2 cells as described in our <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">Protocol page</a>. Figure 4 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.</p>
  
  
  
−
  <div class="d-block" style="width: 60%; margin-left:0%;">
+
  <div class="d-block" style="width: 30%; margin-left:10%;">
 
                           <div class="videoWrapper">
 
                           <div class="videoWrapper">
−
                                                     <iframe title="Optical imaging of Caco-2 cells and fluorescent liposomes functionalized with Anti-HER2 nb  after 1 hour incubation." width="800" height="700" src="https://video.igem.org/videos/embed/f09f1a4d-45fd-40fa-a395-01aed5fa4321?loop=1&amp;autoplay=1&amp;muted=1&amp;title=0&amp;warningTitle=0&amp;controlBar=0&amp;peertubeLink=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
+
                                                     <iframe title="Optical imaging of Caco-2 cells and fluorescent liposomes functionalized with Anti-HER2 nb  after 1 hour incubation." width="800" height="700" src="https://video.igem.org/videos/embed/cfc833c6-1287-405d-a460-bd3da0f31fd5?loop=1&amp;autoplay=1&amp;muted=1&amp;title=0&amp;warningTitle=0&amp;controlBar=0&amp;peertubeLink=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
 
                                                 </div>
 
                                                 </div>
 
                                             </div>
 
                                             </div>
−
                                             <!--
+
                                              
−
                                             <video controls width="250">
+
                                              
−
                                                <source src="https://video.igem.org/w/eKV82ZkfQ2BnXAwkK5vESK"/>
+
−
                                            </video>-->
+
−
 
+
−
                                            <p class="normal centered"><span class="titre-image"><i><b>Figure 4: Optical imaging of Caco-2 cells (brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with Anti-HER2 nb  after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.</b> </i></span></p>
+
  
 +
                                            <p class="normal centered"><span class="titre-image"><i><b>Figure 4:</b>  Optical imaging of Caco2 cells (Brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with Anti-HER2-nb after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.</i></span></p>
  
−
           <p>Qualitatively, this experiment comforted us in the liposomes capabilities to anchor on cancerous cells. However, it does not allow us to ascertain the specificity of the interaction between the liposome and the cancerous cell. Control liposome samples without anti-HER2 nanobodies or competitive assays with soluble extracellular domain of HER2 added in solution will have to be performed.
+
<div>
−
Other simple experiments to characterize this part can be performed by using purified HER2 protein to demonstrate the binding interaction between the anti-HER2 nanobody and the HER2 purified protein.</p>                                 
+
           <p>Qualitatively, this experiment suggests that liposomes are able to anchor on cancerous cells. However, it does not allow us to ascertain the specificity of the interaction between the liposome and the cancerous cell. Control liposome samples without anti-HER2 nanobodies or competitive assays with soluble extracellular domain of HER2 added in solution will have to be performed.</p>                                 
−
                                 
+
  </div>                           
  
 
<h2>Conclusion and Perspectives</h2>
 
<h2>Conclusion and Perspectives</h2>
−
<p>These experiments provide evidence that the Anti-HER2 nb production was successful and that it can be used to promote liposome anchoring on cancerous cells. However, we would recommend performing more experiments to better characterize the anti-HER2 nanobodies and its interaction with HER2 on cancer cells. </p>
+
<p>These experiments provided evidence that the production of the recombinant Anti-HER2-nb was successful. Moreover, preliminary fluorescence microscopy experiments with cultured Caco2 cells suggest that liposome anchoring on cancerous cells is feasible. However, we would recommend performing more experiments to better characterize Anti-HER2-nb and its interaction with HER2 on cancer cells. </p>
−
<p>The construction , the expression and the purification of this Nanobody Anti-HER2 part can be performed in the BSL-1 laboratory and the Characterization with cancer cells in the BSL-2 laboratory. </p>
+
<p>Construction, expression and purification of this Anti-HER2-nb part can be performed in a Biosafety level-1 laboratory and the characterization with cancer cells in a Biosafety level-2 laboratory.</p>
 
<h2>References</h2>
 
<h2>References</h2>
 
<ol>
 
<ol>

Latest revision as of 08:26, 11 October 2023


Anti-HER2 nanobody

Expression and purification of anti-Her2 nanobody for the anchoring to the liposome.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 166
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 518
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
    Illegal NgoMIV site found at 142
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

Figure 1: Anti-HER2 Nanobody Section. "VHH-HER2" corresponds to the sequence of the Anti-HER2 Nanobody.

Toulouse-INSA-UPS 2023 designed this part to functionalize the liposome with anti-HER2 nanobodies for anchoring to cancer cells. To express and purify this nanobody, hereafter called Anti-HER2-nb , we used the E. coli strain BL21(DE3) with the plasmid pET26b_pelB-HER2-tev-stops, provided to us by Adilya Dagkesamanskaya, a researcher at the Toulouse Biotechnology Institute (TBI). This plasmid contains the gene for Anti-HER2-nb fused to a signal peptide gene called pelB leader, along with a His-tag for purification. The signal peptide was fused to Anti-HER2-nb for periplasmic targeting, which promotes disulfide bond formation.

Protein expression and purification

E. coli expression of Anti-HER2-nb was induced using IPTG, and purification was carried out using Cobalt resin (TALON® Metal Affinity Resin). Two separate batches of protein expression and purification were prepared, and both were successful.

Figure 1: SDS-PAGE analysis (15% acrylamide) of protein samples after purification of periplasmic extract and Coomassie Blue staining. Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/ 500).

The expected size of Anti-HER2 nb is approximately 17 kDa. In Figure 1 one can observe a clear band around this size in the extract and flowthrough, demonstrating expression of the nanobody Anti-HER2. The presence of a similar band in the negative control (No IPTG, lane 2, gel 2 in Figure 1) probably reflects an uncontrolled expression of the nanobody due to promoter leakage. Approximately 2 mL of the nanobody at 17.65 µM was produced for each sample.

Characterisation

To test whether liposomes can anchor to cancer cells via Anti-HER2-nb, we prepared fluorescent liposomes with a diameter of 400 nm. These liposomes contain DGS lipids, which allowed us to attach Anti-HER2-nb (See the protocol here). We used HER2-positive colorectal adenocarcinoma cells called Caco-2 cells to test nanobody-mediated liposome anchoring (See the protocol here). Figure 3 shows a microscopy image of adherent and non-adherent Caco-2 cells observed in Brightfield. For educational purposes, we added labels to the image to highlight some features of living eukaryotic cells that can be seen with a regular optical microscope.

Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification.

Fluorescent liposomes were incubated on top of Caco-2 cells as described in our Protocol page. Figure 4 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.

Figure 4: Optical imaging of Caco2 cells (Brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with Anti-HER2-nb after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.

Qualitatively, this experiment suggests that liposomes are able to anchor on cancerous cells. However, it does not allow us to ascertain the specificity of the interaction between the liposome and the cancerous cell. Control liposome samples without anti-HER2 nanobodies or competitive assays with soluble extracellular domain of HER2 added in solution will have to be performed.

Conclusion and Perspectives

These experiments provided evidence that the production of the recombinant Anti-HER2-nb was successful. Moreover, preliminary fluorescence microscopy experiments with cultured Caco2 cells suggest that liposome anchoring on cancerous cells is feasible. However, we would recommend performing more experiments to better characterize Anti-HER2-nb and its interaction with HER2 on cancer cells.

Construction, expression and purification of this Anti-HER2-nb part can be performed in a Biosafety level-1 laboratory and the characterization with cancer cells in a Biosafety level-2 laboratory.

References

  1. [1]Chabrol E, Stojko J, Nicolas A, et al. VHH characterization.Recombinant VHHs: Production, characterization and affinity. Anal Biochem. 2020;589:113491. https://doi:10.1016/j.ab.2019.113491.
  2. [2]Hartmann L, Botzanowski T, Galibert M, et al. VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH. Protein Sci. 2019;28(10):1865-1879. https://doi:10.1002/pro.3712.
  3. [3]Chabrol E, Fagnen C, Landron S, et al. Biochemistry, structure, and cellular internalization of a four nanobody-bearing Fc dimer. Protein Sci. 2021;30(9):1946-1957. https://doi:10.1002/pro.4147.