Part:BBa_K2114998
pelB_aGFPnano
This part contains the anti-GFP nanobody described by Kubala et al. and the pelB leader sequence in the N-terminal section.
Usage and Biology
The discovery of camelid heavy-chain antibodies and their subsequent modification to single-domain antibodies also called nanobodies[1,2] provide researchers with a wide range of tools including labeling methods for imaging, receptor modulation or therapeutic agents. They exhibit a small size of only 15 kDa and their easy structure enables a high-efficiency production in bacterial strains such as E. coli. The anti-GFP nanobody represents an established and well characterized variant of those proteins. In order to directly utilize their functionality, the purification of the over-expressed nanobody provides the advantage of acquiring a highly concentrated protein solution. The BioBrick in the iGEM registry BBa_K929104 contains the full sequence of the anti-GFP nanobody. However, the purification of the nanobody from a bacterial lysate requires its export into the periplasmatic space in order to avoid inclusion bodies and provide an oxidative environment which promotes the formation of the characteristic disulfide bond. The anti-GFP nanobody was provided to us by our supervisor Dr. Maximilian Ulbrich. To avoid the formation of inclusion bodies and increase the yield of purified nanobodies the pelB leader sequence was included at the N-terminal section of the nanobody. This sequence facilitates the export of the protein into the periplasmatic space.
Characterization
1) Expression
2) Binding kinetic
References
1. 2. 3. 4. 5.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 87
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 54
- 1000COMPATIBLE WITH RFC[1000]
| None |