Part:BBa_K3111011

DARPin929

This part encodes DARPin929 – a binding protein specific to HER2 receptor, that is similar to an antibody in terms of efficiency and specificity. However, due to its stability and ability to be expressed in bacteria, this DARPin can be used in a wider variety of applications (1). The part has had its start and stop codons removed for flexible fusion to N or C terminus.

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]

Usage and Biology

Natural Ankyrin Repeat Proteins are versatile binding molecules which can bind to specific targets and subsequently trigger various molecular mechanisms (e.g. enzyme inhibition or protein anchoring) (2,3). Aiming to manipulate the immunologic potential of said repeat proteins for therapeutic applications, in 2003, Plückthun et al. genetically engineered designed DARPins (4). DARPins are small (14-21 kDa) single-domain binding molecules derived from natural ankyrin repeats (5). As natural repeat proteins, DARPins comprised a several structural motifs which stack to form the repeat protein domain (4,6). The assembly of highly expressed, stable and soluble DARPins happened by production of DARPin libraries using ribosome, phage display etc. indicated that these binding molecules had the potential to circumvent the limitations of monoclonal antibodies and empower new therapeutic approaches (4,6).

DARPin929 has been one of the most frequently used to evaluate probes for molecular imaging. It particularly targets human epidermal growth factor 2 (HER2) whose overexpression is associated with breast cancer and gastroesophageal cancer. Has been shown to bind down to nanomolar affinities to its target (7,8). DARPins have been shown to be very stable and show less tendencies for aggregation, unlike single chain variable fragments since they do not possess cysteines.

Functionalisation

BBa_K3111011 has been used along with BBa_K3111021 for initial investigation of binding to SK-BR-3 HER2+ breast adenocarcinoma cells. Moreover, it was fused with BBa_K3111003 and co-expressed with BBa_K3111032 to create the drug delivery vehicle containing cytotoxic cargo (BBa_K3111502).

Structural modelling of DARPin929 fusion to encapsulins

We suspected that fusing DARPin929 to the outer surfaces of T. maritima and M. xanthus encapsulins may result in steric clashes between encapsulin-DARPin dimers, thus impeding or preventing the assembly of the full encapsulin capsules. In order to assess the feasibility of fusing DARPin929 to either encapsulin, we created protein models using PyMol (GUI and manual edits), Discovery Studio (structure quality), GROMACS (energy minimisation of created dimers) and atomium (rebuilding the full encapsulin capsules).

From Figure 1, 2 ... we can see that ... .

As a result we conclude that fusing DARPin929 to the T. maritima encapsulin is more readily feasible. We demonstrate this fusion in BBa_K3111501.

Experimental Results

WIP. See BBa_K3111201, BBa_K3111202, BBa_K3111501, BBa_K3111502 and BBa_K3111502.