Development and characterization of affinity peptides using mRNA display and dot blot method

Protein affinity reagents have aptly gained profound importance as capture reagents and

drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the

cost, labor and time associated with production of antibodies focus has recently changed

towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy

to work with, non-immunogenic, cost effective and amenable to scale up. Even though

researchers have developed several affinity peptides, we are far from compiling library of

peptides that encompasses entire human proteome. My thesis describes high throughput

pipeline that can be used to develop and characterize affinity peptides that bind several

discrete sites on target proteins.

Chapter 2 describes optimization of cell-free protein expression using commercially

available translation systems and well-known leader sequences. Presence of internal

ribosome entry site upstream of coding region allows maximal expression in HeLa cell

lysate whereas translation enhancing elements are best suited for expression in rabbit

reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate

combination ensures maximum protein expression of DNA libraries.

Chapter 3 describes mRNA display selection methodology for developing affinity peptides

for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant

is not sufficient to increase selection pressure for up to three rounds of selection and

increasing number of selection rounds increases probability of finding affinity peptide s.

These studies enhance fundamental understanding of mRNA display and pave the way

for future optimizations to accelerate convergence of in vitro selections.

Chapter 4 describes a high throughput double membrane dot blot system to rapidly

screen, identify and characterize affinity peptides obtained from selection output. I used

dot blot to screen potential affinity peptides from large diversity of previously

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uncharacterized mRNA display selection output. Further characterization of potential

peptides allowed determination of several high affinity peptides from having Kd range 150-

450 nM. Double membrane dot blot is automation amenable, easy and affordable solution

for analyzing selection output and characterizing peptides without ne ed for much

instrumentation.

Together these projects serve as guideline for evolution of cost effective high throughput

pipeline for identification and characterization of affinity peptides.