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	(R585A and R588A) are sufficient to eliminate the heparin binding		(R585A and R588A) are sufficient to eliminate the heparin binding
	affinity of AAV2 (Opie et al. 2003)		affinity of AAV2 (Opie et al. 2003)
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	===Usage and Biology===		===Usage and Biology===
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	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>

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Revision as of 16:23, 29 October 2010

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.

Characterization

Specific targeting of tumor cells was, besides producing recombinant virus particles for therapeutical applications, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010.

For development of targeting strategies against EGF receptor (EGFR) over-expressing cancer cells, exhaustive literature search for engineering the surface of the Adeno-associated virus 2 (AAV2) was performed.

Besides insertion of targeting motifs into the viral protein 1 (VP1) open reading frame (ORF), we designed a method for fusing larger motifs to the N-terminus of VP2. It is expected that these peptides become located on the virus surface either by transit through the pores or by exposure during capsid assembly.

Additionally it is required to knock down the natural tropism of the virus towards its primary receptor heparan sulfate proteoglycan (HSPG) in order to prevent infection of healthy cells (Perabo et al. 2006). The binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity of AAV2 (Opie et al. 2003)

Usage and Biology

Sequence and Features

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