Team:Penn/SurfaceDisplayOverview
From 2012.igem.org
Surface Display and Targeting
![](https://static.igem.org/mediawiki/2012/a/a4/Surfacedispschematic.gif)
Objectives
Our goals were twofold:
- Achieve the first display of DARPin H10-2-G3 on the surface of E. coli and verify that the system targets cancer cells.
- Create a generalized BioBrick surface display platform for other labs and iGEM teams.
Ice Nucleation Protein
We chose to use the Ice Nucleation Protein (Figure 1) as our surface display carrier protein. The Ice Nucleation Protein protein has been used to display enzymes [1], typically for biocatalysis applications (e.g. Edinburgh iGEM 2011). To make the protein a more manageable size, we truncated the protein to the N and C terminal domains only. The C terminal domain is displayed at the cell surface, while the N terminal domain remains in the outer membrane. We sought to apply this system to health/medicine by displaying DARPin H10-2-G3.
![](https://static.igem.org/mediawiki/2012/e/e4/INPNC.png)
Figure 1
Figure 1: Proposed Structure of Ice Nucleation Protein.
[1] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028
DARPin H10-2-G3
The lab of Andreas Plueckthun at ETH Zurich has pioneered the development of Designed Ankyrin Repeat Protein (DARPin) technology. DARPins are engineered antibody-mimetic proteins consisting of 3-5 ankyrin repeat motifs. These proteins have been rationally evolved to high binding affinity with targets through ribosome display. In 2007, the group developed H10-2-G3 [2], a 14.7kDa DARPin evolved to 90pM affinity with the extracellular domain of HER2 (Figure 2).
![](https://static.igem.org/mediawiki/2012/b/b7/DARPingif.gif)
Figure 2
Figure 2: Structure of DARPin-H10-2-G3.
[2] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028