Nanobodies have the potential to act as in vivo reagents, to bind to certain targets for instance, either to block endogenous reactions or to highlight locations in the cell via fluorescence. Therefore, nanobodies are often expressed as fusion proteins. In the case of fusion proteins, are the fused portions always added to the C-terminus of nanobodies to leave the antigen binding site at the N-terminus unobstructed? Or are there any cases reported where proteins have been fused to the N-terminus of nanobodies without obstructing antigen binding?
The N-terminus is on the same side of the nanobody domain as the CDR loops, the C-terminus is on the opposite side. A fusion to the N-terminus does not necessarily need to clash with a bound antigen, but statistically it is more likely to cause problems than a fusion to the C-terminus, especially if you fuse a large domain rather than just a small tag. For an example, look at structure 6H7O (https://www.rcsb.org/3d-view/6H7O/1)
from my own experience, a free N terminus will certainly make nanobody binding more efficient so I would recommend getting rid of whatever fusion protein may have been used to express it
Dear Jürgen, we and others have already reported the fusion of nanobodies with a Nter fusion, notably a Fbox fusion (Elife. 2016 Jul 19;5. pii: e16228. doi: 10.7554/eLife.16228 or Nat Struct Mol Biol. 2011 Dec 11;19(1):117-21. doi: 10.1038/nsmb.2180) without affecting their binding capacity.
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