Knottins provide interesting
structural scaffolds for constrained libraries
The use of nonimmunoglobulin scaffolds, including knottin-based scaffolds,
to engineer novel binding molecules has been reviewed by
Binz et al. in 2005
and by Hosse et al. in 2006.
Sequence randomization, phage-display and amplification-selection
technics allow selection of optimal binders of protein targets.
The cellulose-binding domain of the fungal enzyme cellobiohydrolase
I has been used as a structural scaffold. Although this two-disulfide domain
contains the CSB elementary motif also contained by knottins,
it is not a knottin and does not display the typical "disulfide through disulfide
knot". Seven residues, located on the surface of the
domain, were varied by random mutation of the gene. The repertoire
was cloned for display on filamentous bacteriophage, and selected
for binding to cellulose or alkaline phosphatase. Binders of
cellulose and alkaline phosphatase were isolated
[Smith et al., 1998].
More recently, the two-disulfide Cystine Stablized Beta-sheet motif from a
squash inhibitor (The 'Min-23' peptide) has been used succesfully to select
21 new specific binders on 7 different targets.
Min-23 is one of the smallest known structural scaffold for efficient phage display
and selection [Souriau et al., 2005].
The mRNA display is another approach to identify
peptide sequences involved in macromolecular recognition. A constrained
peptide library has been built using this technic based on the
Knottin EETI-II, where the six residues in the first loop
were randomized. The constrained library was screened against
the natural target of wild-type EETI-II. Selected sequences revealed
minimal consensus sequences of PR(I,L,V)L for the inhibitory
loop of EETI-II and the wild-type sequence, PRILMR, was selected
with the highest frequency [Baggio
et al., 2002].