Part:BBa_K404314

DARPin-E01

[DARPin-E01
BioBrick Nr.	BBa_K404314
RFC standard	RFC 25
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 56
  Illegal XhoI site found at 238
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Binz H.K., Stumpp M.T., 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. (2003), 489 – 503
Kohl A., Binz H.K., Forrer P., Stumpp M.T., Plückthun A., Grütter M.G.Designed to be stable: Crystal structure of a consensus ankyrin repeat protein, PNAS (2003), 1700 – 1705
Steiner D., Forrer P, Plückthun A.Efficient Selection of DARPins with Sub-nanomolar Affinities using SRP Phage Display, J. Mol. Biol. (2008), 1211 – 1227