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

141 Pages·2013·5.58 MB·English

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
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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
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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 .................................................................................. 77
Construction of I-SceI plasmid ............................................................................................... 77
Time-course trial of doxycycline induction ............................................................................ 78
I-SceI expression western blots ............................................................................................. 79
Magnetic bead selections ...................................................................................................... 80
Mock library enrichments ...................................................................................................... 80
Construction of opposite mating type yeast strains .............................................................. 81
3.5 Strains, Plasmids, and Oligonucleotides ........................................................................ 83
3.6 References ..................................................................................................................... 85
Chapter 4 ........................................................................................................................................ 88
Mating Libraries for the Combination of Loop Diversity ............................................................... 88
4.1 Abstract .......................................................................................................................... 88
4.2 Introduction ................................................................................................................... 88
4.3 Results ............................................................................................................................ 92
Fully random NNB and NNY libraries in cassette plasmid ..................................................... 92
An enriched library of FG loops ............................................................................................. 92
Mating full NNB/NNY libraries with Wojcik FG library........................................................... 93
Hamming 2 BC loop library .................................................................................................... 95
Mating H2BC library with Wojcik FG Library .......................................................................... 99
Yeast display of peptidase heterozygotes ........................................................................... 100
4.4 Discussion ..................................................................................................................... 100
Materials and Methods ............................................................................................................ 102
Determining mating efficiency ............................................................................................. 102
Building fully random BC loop libraries into cassette plasmid ............................................ 103
Building Wojcik FG library into yeast display plasmid ......................................................... 104
Building H2BC library ........................................................................................................... 105
Building H2FG library ........................................................................................................... 107
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Mating H2BC library with Wojcik FG library ........................................................................ 108
Building library strains ......................................................................................................... 108
Flow cytometry .................................................................................................................... 109
Testing yeast display in peptidase heterozygotes ............................................................... 109
4.6 Strains, Plasmids, and Oligonucleotides ...................................................................... 110
4.7 References ................................................................................................................... 112
Chapter 5 ...................................................................................................................................... 113
Future Directions ......................................................................................................................... 113
5.1 Abstract ........................................................................................................................ 113
5.2 Discussion ..................................................................................................................... 113
New libraries ........................................................................................................................ 113
Characterization of Binding Hits .......................................................................................... 113
Re-run old libraries with new labeling ................................................................................. 114
Specificity and additional SH2 domains ............................................................................... 114
Improved Mutagenic Systems ............................................................................................. 115
5.3 References ................................................................................................................... 116
Appendix 1: A word about serial processes affecting library complexity………………………………… 118
Appendix 2: Programs for Mining Sequence Data………………………………………………………………….. 120

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List of Figures

Figure 1-1 10Fn3 domain shown from two angles. 4
Figure 1-2 Schematic of yeast cell surface display 5
Figure 1-3 Homologous recombination of mutagenic cassette into
target gene stimulated by endonuclease 12
Figure 1-4 Heritable recombination system for affinity maturation
of protein binders 14
Figure 2-1 Expression and purification of GST-SH2 fusion protein. 26
Figure 2-2 HA4 monobodies expressed on the surface of yeast give
double-positive signal in flow cytometry. 30
Figure 2-3 BC loop residues targeted for mutagenesis. 31
Figure 2-4 Gap-repair mutagenesis for construction of BC loop libraries 32
Figure 2-5 Population sequencing of BC loop coding regions 38
Figure 2-6 FACS enrichment of mixed-length NNB BC loop library 39
Figure 2-7 FACS enrichment of mixed-length NNY BC loop library 39
Figure 2-8 Construction of mutagenic BC cassettes by
oligonucleotide extension PCR. 52
Figure 3-1 A three plasmid system for in vivo mutagenesis 61
Figure 3-2 BC placeholder in pHRS-YD1B has diagnostic features. 63
Figure 3-3 Chromosomal replacement of pep4::HIS3 by
homologous recombination. 65
Figure 3-4 Gain of KanMX, loss of HIS3 marker in yeast transformants. 66
Figure 3-5 Cassette designed to target a monobody loop for mutagenesis. 67
Figure 3-6 A PCR assay to detect homologous recombination of the BC loop. 68
Figure 3-7 PCR assay can distinguish placeholder BC from HA4 BC. 69
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Figure 3-8 Increased homologous recombination with longer
doxycycline induction. 70
Figure 3-9 Western blot analysis of I-SceI expression under reverse tet
transactivator system. 72
Figure 3-10 Detection and Enrichment of Recombinants from Mock Libraries. 73
Figure 3-11 Library size determination by recombination and enrichment. 74
Figure 3-12 Flow cytometry shows enrichment of cells displaying non-truncated
monobody on surface. 75
Figure 3-13 Galactose induction of HO endonuclease results in a, α,
and diploid cells. 75
Figure 4-1 Fully random BC x Wojcik FG, after one round magnetic beads. 94
Figure 4-2 NNB x Wojcik FG after 1 round of beads and 1 round of FACS. 95
Figure 4-3 Mutational scheme for H2BC library 95
Figure 4-4 Population sequencing of H2BC library. 96
Figure 4-5 FITC labeling scheme inadequate for high cell density 97
Figure 4-6 An improved method for labeling yeast display. 98
Figure 4-7 H2BCgap 99
Figure 4-8 H2BCvitro 99
Figure 4-9 Diploids that are heterozygous for yeast display knock-outs
can still express monobodies on the cell surface at
high copy-number. 100
Figure 4-10 Hamming Shells 101
Figure 4-11 First step of random BC cassette synthesis. 103
Figure 5-1 ITC data for the binding of HA4 10Fn3 to GST-SH2. 114

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List of Tables

Table 2-1. Magnetic bead binding capacities. 27
Table 2-2 Magnetic bead enrichments of mock libraries in cell frequencies. 28
Table 2-3 Magnetic bead enrichments of mock libraries in absolute numbers 29
Table 2-4 BC residues sequence from colony PCR of 8x NNB library. 33
Table 2-5 Observed and expected residue frequencies in NNB library 34
Table 2-6 Degenerate codons and their corresponding residue frequencies 36
Table 2-7 Strains used in this study 55
Table 2-8 Plasmids used in this study 56
Table 2-9 Oligonucleotides used in this study 56
Table 3-1 Strains used in this study 83
Table 3-2 Plasmids used in this study 83
Table 3-3 Oligonucleotides used in this study 84
Table 4-1 Theoretical “Hamming 2” (H ≤ 2) library complexities
for different types of diversity 91
Table 4-2 Fully Random NNB/NNY Library Complexity 92
Table 4-3 Sequencing of FG loops found in Wojcik library. 93
Table 4-4 Strains used in this study 110
Table 4-5 Plasmids used in this study 110
Table 4-6 Oligonucleotides used in this study 111

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