1. The study aimed to identify peptide inhibitors of the CD33 receptor, which is associated with increased Alzheimer's risk, using phage display. 2. Researchers purified the CD33 protein and screened a phage library across two rounds, enriching phages with higher CD33 affinity. 3. Analysis found the library was enhanced over 1,000-fold, suggesting phage display is viable for discovering antagonistic CD33 ligands to potentially lower Alzheimer's risk.

1. An unbiased approach to discovering peptide inhibitors
of Sialic acid binding CD33 receptor
Sarah E. Bailey, Steven Estus, Luke H. Bradley
Sanders Brown Center on Aging, University of Kentucky, Lexington, KY
Introduction
CD33 is a sialic acid-binding cell surface receptor found on
microglia in the brain. Upon binding to sialic acid, CD33 acts to
inhibit microglial activation, leading to an accumulation of amyloid
beta protein (Aβ) in the brain and an increased risk of Alzheimer’s
disease. Given the risk associated with the CD33 receptor, the
protein is a relevant candidate target for pharmacological agents
that may act as competitive inhibitors for the sialic acid binding
site. Our overarching hypothesis is that competitive inhibition of
the CD33 binding site constitutes a valuable pharmacological
approach to inhibit CD33 actions and thereby lower the risk of
Alzheimer’s disease. Since robust pharmacologic agents have been
identified through the phage display technique (1), we chose to
use this approach. In this project, we utilized the phage display
technique to identify peptides that bind CD33 to discover potential
CD33 antagonists. Recombinant soluble CD33 with an engineered
His-6 tag was purified from cell media via Ni-NTA affinity. A 12-
mer phage library was then screened against the purified CD33. In
two cycles of biopanning, eluting and amplifying, the library was
enhanced, with the remaining peptides having a significantly
higher affinity for the CD33 protein than the original library. Our
experiment suggests that phage display is a viable option to
screen for peptides which may serve as antagonistic CD33 ligands.
Methods Results
Significance of competitive
inhibition in CD33
Figure 1. Uninhibited, the CD33 receptor binds to sialic acid, inhibiting microglial
activation ad causing a build up of amyloid beta protein in the brain. In a phage
display, a diverse library of bacteriophage display a multitude of varying peptides that
may act as potential competitive inhibitors for sialic acid. This would block the
pathway that leads to microglial inhibition and decreasing the amount of amyloid beta
protein produced.
Figure 2. Western blot testing of various amounts of Ni-NTA beads loaded with CD33 soluble protein
compared with a positive control sample (U937 cells) and a negative control (media from untransfected
CHO-K1 cells) confirm the presence of the CD33 soluble protein on the beads. Once it was confirmed
that the protein had bound to the affinity beads, it was possible to proceed to phase 2 of the
experiment.
Transfection
• Transfection of recombinant CD33 plasmid engineered with His-6 tag into CHO-K1
cells
Protein
Capture
• Capture of recombinant CD33 soluble protein from media via NI-NTA affinity beads
Testing
• Proof of target protein presence via Western Blotting against control
Phase 1: Collecting CD33 protein Round 1 Round 2
Figure 3. The methodology behind in vitro phage display screening over a target protein. A
phage library containing 1x10^12 different twelve amino acid peptides was screened during the
biopanning process over CD33 soluble protein bound to Ni-NTA affinity beads via His-6 tag binding. A
series of washes were then carried out to remove phage that were not tightly bound to the CD33
protein. Specifically bound phage were eluted off the beads with a low pH buffer and were amplified in
E. coli to rebuild the phage library. For this experiment, the process was repeated twice as time
allowed.
Figure 4. Titering plates from selection rounds one and two of the phage display.
Mkr + ctrl 15 uL 10 uL -ctrl
Mkr
Phase 2: Phage display
Starting
Diversity
Flow Through Elution Total
Enhancement
Pre-Screen 1013 107 1012 10
Round 1 1012 1010 106 107
Round 2 1010 1010 106 1011
Table 1. Diversity and enhancement for each of the two rounds of biopanning completed.
A prescreen of the phage library over naïve beads enhanced the started diversity of 1013
to 1012. The first round of biopanning brought the total enhancement to 107 and the final
round of biopanning ended with a phage library that had been enhanced 1011 fold.
Conclusions
Future Directions
1. CD33 soluble protein is viable as a candidate target protein to pursue in
phage display screening as this experiment suggests that it successfully
binds peptides from the phage library with increasing affinity as the
number of biopanning rounds increases.
2. Further rounds of biopanning and amplification could further enhance
the specificity of the peptide pool and select for ligands with a higher
affinity for CD33
Further rounds of
biopanning and
amplification
Sequencing of
peptide combinations
with highest affinity
for CD33 protein
Sequence analysis to
interpret common
binding patterns
with pharmacological
agents
Acknowledgments
The authors acknowledge Sabita Dumre and Jim Simpson for lab assistance as well as
Sanders-Brown Center on Aging and the Howard Hughes Medical Institute for the
funding and support that made this research project possible.
References
1. Nixon, A. E., Sexton, D. J., & Ladner, R. C. (2014). Drugs derived from phage display: From candidate
identification to clinical practice. mAbs, 6(1), 73–85. http://doi.org/10.4161/mabs.27240
2. Malik, M., Simpson, J. F., Parikh I, Wilfred, B. R., Fardo, D. W., Nelson, P. T. & Estus, S. (2013) CD33 Alzheimer's
risk-altering polymorphism, CD33 expression, and exon 2 splicing. J Neurosci. 33 (33), 13320-5.
http://doi.org/10.1523/JNEUROSCI.1224-13.2013
Flow
Through
Elution
Amplification