Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Robert Garcea

Second Advisor

Leslie Leinwand

Third Advisor

Larry Gold

Fourth Advisor

Norman Pace

Fifth Advisor

Marvin Caruthers

Abstract

Proteins that bind with high-affinity to cellular targets can be useful therapeutic treatments. High-throughput affinity screening of large protein libraries is often more successful at discovering novel high-affinity proteins than rational-design approaches. Display techniques such and phage and yeast display are commonly used in this screening process. However, bacteria and yeast cells can misfold or otherwise inappropriately express mammalian and human proteins due to differences in codon usage, protein folding machinery and post-translational modifications. Therefore, a display system that is entirely based in human cells could aid in the discovery of novel, high-affinity proteins. No such system has been described to date without major limitations.

This thesis describes the development of a novel protein affinity selection system. This system entails the use of the human Herpes Simplex Virus 1 (HSV-1) as a particle for protein display. In this system, genes encoding proteins to be displayed are inserted into the HSV-1 chromosome via a novel recombination system. The viruses produced display the encoded proteins on their surface fused to the C-terminus of HSV-1 glycoprotein C. Large libraries of protein-displaying viruses are then subjected to a novel, competitive-infection selection procedure. Through Darwinian selection, viruses displaying proteins with high binding affinity for cell-surface targets are enriched over viruses displaying proteins with lower binding affinity. Proof-of-principle testing has shown this system can select for a gene encoding a high-affinity protein that is outnumbered 1:105 by genes encoding lower-affinity proteins in a plasmid DNA library. Further testing of this system, using a mutant library of genes encoding the CX3CL1 chemokine, identified novel CX3CL1 mutants that may bind to the CX3CR1 receptor with higher affinity than wild-type CX3CL1. This system was also used to screen a B-cell antibody library from an HIV-infected individual. This system identified several novel antibodies that are very similar to high-affinity antibodies previously discovered from the same library. The results of this work show that this novel selection system may be a widely-applicable tool for the discovery of high-affinity human and mammalian proteins.

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