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Building a Genetic System in Yeast to Search for High Affinity Proteins in Sequence Space

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Building a Genetic System in Yeast to Search for High Affinity Proteins in Sequence Space Building a Genetic System in Yeast to Search for High Affinity Proteins in Sequence Space Matthew Douglas Merguerian Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2013 Matthew Douglas Merguerian All rights reserved ABSTRACT Building a Genetic System in Yeast to Search for High Affinity Proteins in Sequence Space Matthew Douglas Merguerian Binding proteins (both natural and man-made) have the ability to bind tightly and specifically with small molecules and other biopolymers. Binding proteins can function as human therapeutics, diagnostics, and as tools for scientific research. Given the wide range of potential applications, there is great interest in both academia and industry to develop methods for discovering novel binders. An important step in discovering new binders is called affinity maturation, when an initial hit that shows some ability to bind the target is further mutated in additional steps to improve binding affinity, specificity, solubility, pharmacokinetic profile. Ideally, the methods for affinity maturation would allow for cookbook protocols, be successful for arbitrary targets of interest, and be minimally resource intensive. Although traditional methods for affinity maturation have had some stunning successes over the past, the state of the field is still far from this ideal. In Chapter 1, I discuss the current state of the protein engineering field. In Chapter 2, I discuss the use of phenotypic selection for yeast-display protein binders, and test these systems on simple loop libraries.. In Chapter 3, I construct a genetic system in yeast that can mutate a protein loop via homologous recombination, and test its recombination function. In Chapter 4, I mate libraries that target two different loops, and run FACS on the combinatorial libraries. In Chapter 5, I discuss future directions for the project. Table of Contents List of Figures v List of Tables vii List of Abbreviations viii Acknowledgements xi Dedication xii Chapter 1 .......................................................................................................................................... 1 Searching for functional protein binders in sequence space........................................................... 1 1.1 Abstract ............................................................................................................................ 1 1.2 Binding proteins ............................................................................................................... 1 1.3 Yeast display ..................................................................................................................... 4 1.4 Directed evolution of proteins ......................................................................................... 7 1.5 Affinity maturation........................................................................................................... 8 1.6 SH2 domains .................................................................................................................... 9 1.7 Harnessing the power of combinatorial mixing ............................................................. 11 1.8 A model system for investigating affinity maturation ................................................... 13 1.9 References ..................................................................................................................... 15 Chapter 2 ........................................................................................................................................ 23 Cconstruction of BC loop libraries and Phenotypic Selection ........................................................ 23 2.1 Abstract .............................................................................................................................. 23 2.2 Introduction ................................................................................................................... 23 2.3 Results ............................................................................................................................ 25 Protein Expression ................................................................................................................. 25 Magnetic Bead Selections ...................................................................................................... 27 Flow cytometry of HA4 .......................................................................................................... 29 Construction of BC loop libraries ........................................................................................... 30 FACS Enrichment on Mixed-Length Libraries ......................................................................... 39 2.4 Discussion ....................................................................................................................... 40 2.5 Materials and Methods .................................................................................................. 42 i General molecular biology ..................................................................................................... 42 Media and buffer recipes ....................................................................................................... 43 DNA Ligation .......................................................................................................................... 44 Making polyacrylamide gels for SDS-PAGE ............................................................................ 45 Expression of GST-SH2 fusion protein ................................................................................... 45 Purification of GST-SH2 fusion protein .................................................................................. 46 Biotinylation of GST-SH2 ........................................................................................................ 47 Galactose Induction of Yeast Display ..................................................................................... 48 Magnetic Bead Capacity Experiment ..................................................................................... 48 Magnetic Bead Mock Library Enrichment .............................................................................. 49 Flow Cytometry of ysEBYHA4 ................................................................................................ 50 Yeast Display Plasmid Alterations .......................................................................................... 50 Construction of mutagenic BC cassettes ............................................................................... 52 FACS sorting of libraries ......................................................................................................... 53 Yeast Transformation ............................................................................................................. 53 Yeast Colony PCR ................................................................................................................... 54 2.6 Strains, Plasmids, and Oligonucleotides ........................................................................ 55 2.7 References ..................................................................................................................... 57 Chapter 3 ........................................................................................................................................ 60 A recombination system for in vivo mutagenesis .......................................................................... 60 3.1 Abstract .......................................................................................................................... 60 3.2 Introduction ................................................................................................................... 60 3.3 Results and Discussion ................................................................................................... 62 Construction of diagnostic yeast display plasmid .................................................................. 62 Plasmids for inducible expression of I-SceI endonuclease and DSB’s.................................... 63 Replacing HIS3 with KanMX in the genome of EBY100 ......................................................... 64 Plasmids containing mutagenic cassettes flanked by I-SceI cut sites .................................... 66 Assays for I-SceI induced homologous recombination .......................................................... 67 Doxycycline timecourse for induction of homologous recombination ................................. 70 Western blot of I-SceI expression .......................................................................................... 71 Mock libraries to determine recombination efficiency ......................................................... 72 ii Mating type switch in ysMDM100 to create mating partner ................................................ 75 3.4 Materials and Methods .................................................................................................. 76 Construction of yeast display plasmids .................................................................................. 76 Construction of cassette plasmids ......................................................................................... 76 Replacing HIS3 in EBY100 with KanMX .................................................................................
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