Difference between revisions of "Part:BBa K4768002"
 
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<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>
+
<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>
  
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<h2>Construction expression and purification</h2>
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<h2>Protein expression and purification </h2>
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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>
+
<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>
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<p>Two separate batches of protein expression and purification were performed in duplicate, and both were successful.  
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</p>
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             <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)
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             <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>
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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 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>
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<h3>Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells</h3>
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<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>
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<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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             <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>
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             <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.  
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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>
 +
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>
  
  
  
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                                            <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>
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                                            <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>
  
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           <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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           <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>                                 
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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 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>
+
<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>
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<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>
+
<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.