Part:BBa_K4768002

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

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Construction expression and purification

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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Characterisation

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

400 nm fluorescent liposomes were prepared and coated with anti-HER2 nb, see Protocol page. Colorectal adenocarcinoma cells Caco-2, which are HER2-positive, were used to test the anchoring of liposomes by Anti-HER2 nb see Protocol page. Figure 1 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 3 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.

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.

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.

Conclusion and Perspectives

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References

1. article 1 xxxxxxxx
2. article 2 xxxxxxx