Part:BBa_K3117046

scFv bispecific antibody against GPA33 and CD3 codon optimized for E.coli

The part BBa_K3117026 is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (BBa_K3117016). This part is codon optimized for the expression in E.coli.

Usage and Biology

By connecting the variable regions of the heavy and light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (BBa_K3117028), the scFv retains it's 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_K3117037), they bind covalently (Hatlem et al., 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag. The sequence contains a C-terminal His-Tag (BBa_K3117005) for easy purification and detection. Secretion of the protein into the perisplasm is ensured by the pelB sequence leader (BBa_K3117012). When the protein passes the membrane, the leader segment is cleaved off.

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.

Sequence and Features

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]

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.