abstract: 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 ii 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. / Dissertation/Thesis / Masters Thesis Biochemistry 2014

Synthesizing proteins for use in therapeutics is restrained by, in part, contaminants in in vivo expression systems and limited production capacity of in vitro systems. Cell free expression (CFE) systems have emerged as a potential alternative for protein expression because of the inherently lower contents of contaminants, and their flexible modular design that allows the addition of factors that aid in synthesis of complex products. Here, we investigate and establish an in-house Escherichia coli-based cell free protein synthesis (CFPS) system, explore different CFPS commercial kits, develop assays to test performance of these systems and identify potential rules that dictate expression levels. Using CFE, we were able to test different vectors and conditions of system, as well as scale-up protein synthesis reactions. In conclusion, this work shows that CFPS is a functional and easy-to-use platform and can potentially meet the requirements for the synthesis of therapeutics.