High-Throughput Screening of Yeast Display Libraries for the Development of Matrix Metalloproteinase Inhibitors
metalloproteinases (MMPs) have long been considered promising therapeutic targets to
understand and combat cancer development and progression. Given the similarity of their
structures and enzymatic functions, but the considerable variation of their
pro-tumorigenic or tumorigenic suppressive effects, small molecules have proven
unsuccessful as therapeutics, and more specific i... read morenhibitors are required. Of the
23-member family of human MMPs, strides have been made in the development of specific
inhibitors against MMP-2, MMP-9, and MMP-14 using protein-based affinity reagents,
suggesting that the specificity of protein binders will enable improved targeting of
these proteinases. In this work, overall goal is the development of improved strategies
for identifying MMP inhibitors. We isolate and examine single chain variable fragment
(scFv) binders from synthetic antibody libraries in an attempt to isolate cross-reactive
inhibitors against murine and human isoforms of MMP-9 as a proof of concept for the
generation of novel yeast display libraries in the support of protein-small molecule
hybrid (PSMH) development. We further present a rapid, display inhibitor assay to better
examine the location effect of binding on enzymatic activity, to aid characterization
and the advancement of binders to become PSMHs. Finally, we demonstrate the challenge of
multiplexed magnetic bead sorting, meant to simultaneously isolate specific binders to a
multitude of antigens from a single protein display library, to expedite the process of
high-throughput screening and affinity reagent
Thesis (M.S.)--Tufts University, 2017.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: James Van Deventer.
Committee: Nikhil Nair, and Qiaobing Xu.
Keyword: Engineering.read less