Development and application of new platform technologies for protein engineering [electronic resource]

Protein engineers rely heavily on directed evolution, a powerful method that iterates rounds of library mutagenesis and screening to identify proteins with desirable properties. In a directed evolution experiment, randomly generated protein libraries are mined for variants with desirable characteristics, such as high affinity binding to a target of interest, stability, fluorescence, or enzymatic activity. Resulted proteins with an improved function are applied in clinic as novel therapeutic/diagnostic agents or in industry as advanced catalysts. In this dissertation, I demonstrate development and applications of novel platform technologies involved in the key steps for successful combinatorial protein engineering: 1) a sophisticated library construction and 2) a reliable, high-throughput screening of a large-sized library. First, I introduce an improved strategy of yeast surface display, a well-established technology for library construction to analyze and engineer proteins. Streamlining the conventional yeast surface display strategies where displayed-protein variants are expressed as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein, the new yeast surface display platform utilizes both the N- and C-termini of Aga2p to co-express two heterologous proteins. I show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface by allowing an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. Moreover, this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast. Next, I describe the development of a new screening platform technology, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction), and its applications for engineering protein-protein binding interaction. μSCALE is a multi-purpose platform technology capable of interrogating a dense array of millions of spatially segregated single cells or their protein products within a time frame of minutes. A key feature of μSCALE is the ability to isolate target cells post analysis from the microcapillary array using a precise laser-based extraction technique. I first highlight an application of the μSCALE platform in isolation of a novel protein binder from a naïve antibody library, which shows this platform enables screening a rare protein binder from a stringent library condition. Then I demonstrate the use of the µSCALE platform for affinity maturation of a protein binding interaction against a clinical cancer target. Within a significantly facilitated time period, two iterative rounds of library generation and screening resulted in engineered variants with a 50-fold improvement in binding interaction, highlighting the use of µSCALE as a new tool for directed evolution. In addition, this thesis contains the study on cellular internalization of a functional fragment of human hepatocyte growth factor (HGF). I first demonstrate that a natural splicing fragment of HGF, NK1, efficiently enters cells with its highly cationic surface charge density. Then in-depth mechanism study for the NK1 internalization is followed, which shows NK1 internalizes into mammalian cells by using its ability to bind particular surface receptors such as heparan sulfate proteoglycans and c-Met. Based on the structural and functional mechanism of the NK1 internalization, I propose a strategy for enhanced angiogenesis by using a cell-penetrating, agonistic NK1 protein to deliver plasmid DNA that encodes an angiogenic factor. This strategy may possibly elongate the duration of therapeutic angiogenic signals at an ischemic site.

Keywords: Bioengineering.
Advisor: Cochran Jennifer R. primary advisor. Thesis advisor
Endy Andrew D. advisor. Thesis advisor
Graves Edward (Edward Elliot)