Difference between revisions of "Part:BBa K2114998"

Revision as of 17:35, 12 October 2016

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. 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. The nanobody can be expressed in standard expression vectors such as pGEX or pET in an appropriate bacterial strain.

Characterization

Bacterial expression

Analysis of the purified pelB anti-GFP nanobody by SDS-PAGE

The anti-GFP nanobody can be expressed in pET303 including a C-terminal 10xHis-tag in the E. coli strain BL21 after induction with IPTG for 3 hours at 37 °C. The lysate was purified using nickel columns and analysed by SDS-PAGE and coomassie staining to verifiy the expected size of 17 kDa.

Binding kinetics
The dissociation constant KD octet Red

Binding kinetic of purified anti-GFP nanobody to 10xHisGFP

References

1. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–8 (1993).
2. Muyldermans, S. & Lauwereys, M. Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies. J. Mol. Recognit. 12, 131–140 (1999).
3.
4.

5.

Sequence and Features