(13e) Engineering of High Affinity Binding Peptides Using N-Terminal Bacterial Display

Display library technologies play a central role in protein
engineering applications, from drug discovery to biomineralization.  Bacterial
display holds the potential to expedite the process of isolating desired
binding proteins, but has been slow to mature owing to technical limitations. 
One such limitation is the inability to display passengers efficiently as N-terminal
fusions.  To address this issue, a large constrained peptide library (X₂C(X)₇CX₂)
(10¹⁰ variants) was displayed via the N-terminus of a circularly
permuted variant of outer membrane protein X (OmpX) in E. coli.  For
comparison, using native OmpX protein an otherwise identical peptide library
was constructed within an extracellular loop as a sandwich fusion.  To evaluate
the two display platforms, each library was screened against streptavidin as a
model target using sequential magnetic and fluorescence activated cell
sorting.  Individual binding clones were obtained in just two to three days.  A
common three-residue streptavidin binding motif, HPQ, and a similar motif, HPM,
were isolated using both the sandwich fusion and N-terminal display scaffolds. 
The N-terminal display format yielded a unique, high affinity motif, CGWMYYXEC,
having six consensus residues.  One of the isolated peptides, expressed as a
terminal fusion to a monomeric fluorescent protein, possessed a dissociation
rate constant of 2x10^-3 s^-1, a value twenty fold improved
relative to the best clone with an HPQ motif.  In addition to displaying
peptides that allow for single disulfide constrained loops, peptide scaffolds
that incorporate multiple cysteines have been used, such as an α-conotoxin
and cyclotide scaffold.  Within these scaffolds libraries were created with
select residues randomized.  Both libraries were successfully screened for
peptides binding to streptavidin, and strong consensus sequences emerged.  The
peptide display libraries were also used to isolate sequences that bind
specifically to pharmaceutically interesting proteins, such as VEGF.  To our
knowledge, this is the first successful demonstration of an N-terminal
bacterial display peptide library using a circularly permuted transmembrane
protein, and the first successful use of libraries incorporating a cyclotide
scaffold.  This novel bacterial display platform offers new opportunities for
investigating protein-peptide interactions as well as a means to rapidly
isolate and engineer high-affinity, specific protein binding peptides.