Difference between revisions of "Part:BBa K4768002"
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<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 Anti-HER2 nanobodies (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 decorate the liposome for anchoring to cancer cells. To express and purify these Anti-HER2 Nanobodies (Anti-HER2 nb), we used the <i>E. coli</i> strain BL21(DE3), which contained the plasmid pET26b_pelB-HER2-tev-stops, provided to us by Adilya Dagkesamanskaya, a researcher at the Toulouse Biotechnology Institute (TBI). This plasmid contained the gene for the Anti-HER2 Nanobody fused to a signal peptide gene called pelB leader, along with a Histag for purification. The signal peptide was fused to the Anti-HER2 nb with the aim of achieving periplasmic expression.</p>
  
 
<h2>Construction expression and purification</h2>
 
<h2>Construction expression and purification</h2>
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<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.  
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<p>The expression of this Anti-HER2 nb part was induced using IPTG, and purification was carried out using Cobalt resin (TALON® Metal Affinity Resin). </p>
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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>Two separate batches of protein expression and purification were performed in duplicate, and both were successful.  
 
</p>
 
</p>
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<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 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>
  
  
 
<h2>Characterisation</h2>
 
<h2>Characterisation</h2>
 
<h3>Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells</h3>
 
<h3>Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells</h3>
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<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>
+
<p>To test how liposomes anchor to cancer cells using Anti-HER2 Nanobodies, we prepared standardized fluorescent liposomes with a diameter of 400 nm. These liposomes contain DGS lipids, which allow us to coat the liposomes with the Anti-HER2 Nanobodies. <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. We used HER2-positive colorectal adenocarcinoma cells called Caco-2 cells to test how well the liposomes could attach with the help of the Anti-HER2 Nanobodies <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. Figure 3 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>
 
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<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>
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<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 an animated gif picture with the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.</p>
  
  
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           <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.
+
           <p>Qualitatively, this experiment seems to show 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>                                 
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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>                                 
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<h2>Conclusion and Perspectives</h2>
 
<h2>Conclusion and Perspectives</h2>
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<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 provide evidence that the Anti-HER2 nb production was successful and that it seems possible to use it 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>
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<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>The construction , the expression and the purification of this Nanobody Anti-HER2 part can be performed in the Biosafety level-1 laboratory and the characterization with cancer cells in the Biosafety level-2 laboratory.</p>
 
<h2>References</h2>
 
<h2>References</h2>
 
<ol>
 
<ol>

Revision as of 14:24, 8 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 part

Toulouse-INSA-UPS 2023 designed this part to decorate the liposome for anchoring to cancer cells. To express and purify these Anti-HER2 Nanobodies (Anti-HER2 nb), we used the E. coli strain BL21(DE3), which contained the plasmid pET26b_pelB-HER2-tev-stops, provided to us by Adilya Dagkesamanskaya, a researcher at the Toulouse Biotechnology Institute (TBI). This plasmid contained the gene for the Anti-HER2 Nanobody fused to a signal peptide gene called pelB leader, along with a Histag for purification. The signal peptide was fused to the Anti-HER2 nb with the aim of achieving periplasmic expression.

Construction expression and purification

The expression of this Anti-HER2 nb part 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 performed in duplicate, and both were successful.

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)

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.

Characterisation

Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells

To test how liposomes anchor to cancer cells using Anti-HER2 Nanobodies, we prepared standardized fluorescent liposomes with a diameter of 400 nm. These liposomes contain DGS lipids, which allow us to coat the liposomes with the Anti-HER2 Nanobodies. see Protocol page. We used HER2-positive colorectal adenocarcinoma cells called Caco-2 cells to test how well the liposomes could attach with the help of the Anti-HER2 Nanobodies see Protocol page. Figure 3 shows a microscopy image of adherent and non-adherent Caco-2 cells 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.

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 an animated gif picture with 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 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.

Qualitatively, this experiment seems to show 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 provide evidence that the Anti-HER2 nb production was successful and that it seems possible to use it 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.

The construction , the expression and the purification of this Nanobody Anti-HER2 part can be performed in the Biosafety level-1 laboratory and the characterization with cancer cells in the 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.