Team:UCL/Basic Part

Best Basic Part

BBa_K3111011 - DARPin929

Designed Ankyrin Repeat Proteins (DARPins) are small (14-21 kDa) single-domain binding molecules which can bind to specific targets and subsequently trigger various molecular mechanisms (e.g. enzyme inhibition or protein anchoring) (1,2). New DARPins can be created via mutagenesis and phase display technologies, which enables the selection of highly stable, soluble, easily-expressed and efficient versions. This indicates that these binding molecules have the potential to circumvent the limitations of monoclonal antibodies and empower new therapeutic approaches (3).

In attempt to explore their therapeutic potential, we used DARPin929 (BBa_K3111011) as a binding molecule for our drug delivery platform. DARPin929 was designed to target human epidermal growth factor 2 (HER2) receptor, whose overexpression is associated with breast cancer and gastroesophageal cancer (4). To observe this, we fused the DARPin with mScarlet, a red fluorescent protein, confirming binding with confocal microscopy and flow cytometry. We demonstrated that DARPin929 retains its specificity when fused on the C-terminus of another protein in BBa_K3111201 and BBa_K3111202, as structural modeling showed concerns on the binding site getting buried. This property was then used to join DARPin929 to the outer shell of T. maritima encapsulin, where it was shown to retain specificity to HER2 receptor.

References

  1. Janeway CA, Medzhitov R. Innate Immune Recognition. Annu Rev Immunol. 1 de abril de 2002;20(1):197-216.
  2. Forrer P, Stumpp MT, Binz HK, Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 27 de marzo de 2003;539(1):2-6.
  3. Binz HK, Stumpp MT, Forrer P, Amstutz P, Plückthun A. Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins. J Mol Biol. 12 de septiembre de 2003;332(2):489-503.
  4. 21. Camacho-Leal MP, Sciortino M, Cabodi S. ErbB2 Receptor in Breast Cancer: Implications in Cancer Cell Migration, Invasion and Resistance to Targeted Therapy, Breast Cancer - From Biology to Medicine. Phuc Van Pham, IntechOpen.