Functional Characterization of Fibrinolysis-Modulating Proteins Using High Throughput Phage Display

Abstract

The advent of modern high throughput sequencing (HTS) technology has resulted in the resurgence of phage display as a powerful technique in screening peptide and protein libraries for mutations and/or protein fragments with desired properties. As the protein or peptide is covalently tethered to (or “displayed” on) the surface of the bacteriophage, its corresponding genetic material may be rapidly identified and analyzed using DNA sequencing technologies. As the earliest applications of phage display were limited by the available methods (low-throughput Sanger DNA sequencing), repetitive rounds of selection and amplification were required, and only a small portion of the selected phage could be analyzed. Coupling phage display with HTS creates a new high throughput platform, termed high throughput phage display, with which to quickly and without bias screen vast libraries of peptides and proteins for novel binding interactions, stability, and kinetics. High throughput phage display allows characterization of the population of a library of >106 phage prior to and after selection to determine the level of enrichment or depletion of individual clones within the library. In this dissertation, I will highlight the potential of high throughput phage display with two different yet complimentary applications:

Streptococcus pyogenes (GAS) is a pathogenic bacteria that activates the fibrinolytic system via a secreted virulence factor, streptokinase, facilitating GAS dissemination throughout the human host and evasion of the host immune system. A small, bioactive compound that inhibits streptokinase expression was previously developed and validated in a humanized mouse model of GAS septicemia. The mechanism of action and target of the compound within GAS remain unknown. A candidate gene was ruled out using genetic studies so an unbiased biochemical approach to identify GAS proteins involved in its mechanism of action was undertaken. Randomly fragmented GAS genomic DNA was cloned into a phage display vector in order to create a library of phage displaying GAS peptides on their surface. Although the utility of this library was hindered by technical difficulties during preparation, it demonstrated feasibility of constructing large and diverse phage display libraries that could be useful in other applications.

As a serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) regulates the fibrinolytic system by inhibiting the plasminogen activators, tissue-type and urokinase-type plasminogen activator PAI-1 undergoes spontaneous conformational conversion to an inactive, latent state with a half-life of 2 hours. Two previous whole gene mutagenesis studies identified mutations that stabilize the active conformation of PAI-1 but ultimately only characterized the effects of 1.2% of all possible amino acid substitutions on PAI-1 stability. Here we report the construction and screening of a phage display library of PAI-1 variants to examine the mutational landscape with respect to functional stability. Using high throughput phage display, we surveyed 74% of all possible amino acid substitutions within PAI-1 resulting in identification of 1509 substitutions that decrease the functional half-life of PAI-1 and 492 mutations that stabilize the active conformation, including the majority of previously reported mutations. Our approach provides a massively parallel platform to comprehensively analyze the largely unknown mutational landscape of PAI-1 and the effect of single amino acid substitutions on functional stability of PAI-1. This approach could be applied in the future to characterize the clinical significant of all possible human mutations and to generate PAI-1 variants with altered target specificity and the potential to treat human diseases due to serpinopathies.