Ligand Binding

Protein binding

Affibody

Affibodies are small (6 kDa), soluble high-affinity proteins. They are derived from the IgG-binding B domain of the Staphylococcal protein A, which was engineered to specifically bind to certain peptides or proteins. This so-called Z domain consists of an antiparallel three-helix bundle and is advantageous due to its proteolytic and thermodynamic stability, its good folding properties and the ease of production via recombinant bacteria (Nord et al., 1997). Affibodies can be used for example for tumor targeting (Wikman et al., 2004) and diagnostic imaging applications (Orlova et al., 2006; Orlova et al., 2007). The ZEGFR:1907 Affibody was engineered to specifically bind the EGF receptor with an affinity determined to be KD = 2.8 nM (Friedman et al., 2008). The EGF receptor is overexpressed in certain types of tumors, e.g. in breast (Walker & Dearing, 1999), lung (Hirsch et al., 2003) and bladder (Colquhoun & Mellon, 2002) carcinomas, and is therefore a suitable target for cancer imaging or therapeutic applications. Because of their good tumor uptake, and their property to become internalized into the target cells with an efficiency of 19 – 24% within one hour – compared to 45% of the natural ligand EGF - the ZEGFR:1907 Affibody was chosen for therapeutic applications by the Freiburg iGEM Team 2010 (Friedman et al., 2008; Göstring et al., 2010).

Name	Target	Amino acids	Mol. mass [kDa]	BioBrick RFC10	BioBrick RFC25	Team	Reference
Z-EGFR-1907	HER2				BBa_K404302	[http://2010.igem.org/Team:Freiburg_Bioware Freiburg 2010]

Darpin

Natural protein ankyrin repeat (AR) molecules are motifs that can be found commonly in proteins (Bork 1993). These motifs mediate protein-protein interactions suggesting that AR proteins can be used for designing new binding molecules. Design of structural scaffolds with consensus regions and randomized positions of interacting residues leads to improved biophysical characteristics of targeting molecules (Binz et al. 2003) (Kohl et al. 2003).

The repetitive nature of the ankyrin proteins allows modifications in their variable and modular binding surface. Therefore, consensus sequences of natural ankyrin proteins have been used to design novel and stable scaffolds for binding proteins. Designed Ankyrin Repeat Proteins (DARPins) are well expressed, monomeric in solution, thermodynamically stable and have the ability to fold fast. In the publication of (Steiner et al. 2008) screening libraries were created by using the signal recognition particle (SRP) translocation pathway for phage display. The selected DARPin E_01 has very high affinities to the target protein ErbB1 and can be used as a potential targeting molecule for our approach by fusing the DARPin to N-terminal VP proteins. Our designed ankyrin repeat protein consists of three internal binding repeats and the C-and N-terminal capping repeats. Each internal repeat module comprises one beta-turn and two hydrophobic alpha helices. The potential interaction residues are located in the beta-turn and the first alpha helix of the AR-proteins.

Name	Target	Amino acids	Mol. mass [kDa]	BioBrick RFC10	BioBrick RFC25	Team	Reference
DARPin-E01	HER2				BBa_K404302	[http://2010.igem.org/Team:Freiburg_Bioware Freiburg 2010]

Anticalin

The Anticalin technology was developed by Prof. Dr. A. Skerra (TU Munich, Germany) that are based on lipocalins The anticalins availible as BioBricks are the digoxigenin binding anticalin DigA (BBa_K243003) and the fluorescein binding anticalin FluA (BBa_K157004).

Name	Target	Amino acids	Mol. mass [kDa]	BioBrick RFC10	BioBrick RFC25	Team	Reference
DigA	Digoxigenin				BBa_K243003	[http://2010.igem.org/Team:Freiburg_Bioware Freiburg 2010]
FluA	Fluorescein				BBa_K157004	[http://2010.igem.org/Team:Freiburg_Bioware Freiburg 2010]

Small molecule binding

Fluoreszein Digitonin

Sugar binding

• Cellulose Binding