Part:BBa_K3117026:Design

scFv against GPA33 with SpyCatcher codon optimized for CHOs

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

File:Https://2019.igem.org/wiki/images/a/a8/T--FAU Erlangen--Bild Sequencing K2b Results Composite part.png

Figure 1: Induction temperature –standard luminescence value

The sequence contains a C-terminal His-tag (BBa_K3117005) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (BBa_K3117006), which directs the produced protein into the secretory pathway. When the protein passes the membrane, the leader segment is cleaved off. By connecting the variable regions of the heavy and the light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (BBa_K3117028), the scFv retains its antigen-binding ability and is much smaller than a conventional antibody.

The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacteria Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyTag (BBa_K3117015), they bind covalently (Hatlem, Trunk, Linke, & Leo, 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag.

Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.

Source

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References

1. Hatlem, D., Trunk, T., Linke, D., & Leo, J. C. (2019). Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. International journal of molecular sciences, 20(9), 2129.

2. Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., . . . Denis, M. G. (2009). KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines. International journal of cancer, 125(12), 2802-2809.

3. Schoene, C., Fierer, J. O., Bennett, S. P., & Howarth, M. (2014). SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme. Angewandte Chemie International Edition, 53(24), 6101-6104.