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{Replace with the Title of Your Dissertation} In vitro evolution of artificial enzymes: method development and applications A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY John Christian Haugner III IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Burckhard Seelig September 2014 © John Christian Haugner III, 2014 Acknowledgements There are many people past and present in my thesis lab I’d like to acknowledge for all their support. First and foremost I thank my advisor, Dr. Burckhard Seelig, for guiding me in my research and challenging me to improve. I thank Dr. Misha Golynskiy for being a wonderful friend and colleague; we spent a few long years together working on those libraries and you always helped lift my spirits when experiments went wrong. I thank Aleardo Morelli who was a true comrade throughout all of our work with mRNA display and ligase 10C. I also thank Dr. Dana Morrone and Michael Lane for all the enlightening discussions we shared about enzyme selections and their input in many of my publications and presentations. Outside my thesis lab, I thank Fa-An (Frank) Chao and the rest of the Veglia lab for their excellent work in solving the structure of ligase 10C. I thank our many collaborators described in chapter 6 who have helped us develop our application for ligase 10C. I’d like to acknowledge the NIH Biotechnology Training Grant (T32 GM08347) for providing funding and broadening my experiences in Biotechnology. I’d also like to acknowledge the Doctoral Dissertation Fellowship for helping fund my final year of research and giving me the opportunity to share my research to a broader audience. Finally, I’d like to thank all the marvelous people of the Biotechnology Institute here at the University of Minnesota. I thank the administrators, faculty and staff for putting together an incredible program for graduate students. I also thank all of my fellow researchers within Gortner Laboratory for sharing not just materials and equipment, but a real sense of collaboration and comradery. i Dedication For my wife, Elizabeth, and to my daughter, Evelyn, who have brought endless joy into my life. ii Abstract Artificial enzymes have the potential to aid in the production of pharmaceuticals and facilitate basic biomedical research. There are two methods for making artificial enzymes: rational design and de novo selection. Rational design utilizes detailed knowledge of enzyme catalysis to design an enzyme active site, and then introduces this active site into a protein. However, due to the limited understanding of protein folding and structure-function relationships this approach is still extremely challenging and far from routine. In contrast, we utilize a directed evolution approach to isolate de novo artificial enzymes from a large library of protein variants by in vitro selection. Each of the trillions of proteins in a library are tested in a single experiment to determine if any have the desired activity. The artificial enzymes are created when the library is made so a high quality library is important for success. My thesis research focuses on two goals: (1) Construct a library built on the robust (β/α)8 barrel enzyme scaffold for future enzyme selections and (2) Characterize a thermostable artificial RNA ligase and develop an application for this enzyme. The (β/α)8 fold is used to catalyze a wide range of chemical reactions in nature. We used this fold to create a library containing > 1014 unique proteins by replacing loops of the catalytic face with randomized codons via PCR. Small sub-libraries were subjected to a protease-based folding selection to improve library quality by enriching for folded sequences. The final folding-enriched library contained > 1012 folded proteins representing an up to 50-fold improvement relative to a control library. These libraries will provide a valuable source of new enzymes for future in vitro selections. The previously generated artificial RNA ligases join 5’-triphosphate RNA to the 3’-hydroxyl of a second RNA substrate; a reaction not observed in nature. However the enzymes were also highly dynamic, which prevented the solving of the protein structure by NMR or X-ray crystallography. A more structured enzyme, called ligase 10C, was isolated by performing the ligase selection at 65°C and its structure was solved revealing a novel primordial fold. Here, we describe the detailed biochemical characterization of ligase 10C. Using a variety of RNA substrates, we also determined how ligation rates iii change with sequence composition revealing an enzyme with broad sequence specificity. We developed a method for the specific ligation and sequencing of 5’-triphosphorylated RNA. These results highlight ligase 10C as an attractive tool for the selective isolation of 5’-triphosphate RNA from a complex mixture, something which is difficult with current methods. iv Table of Contents Acknowledgements .................................................................................................... i Dedication .................................................................................................................. ii Abstract ...................................................................................................................... iii Table of contents ........................................................................................................ v List of tables ............................................................................................................... ix List of figures ............................................................................................................. x Chapter 1: Introduction ………………………………………….….. 1 1.1 Thesis overview…………………………………………………………….. 1 1.2 Significance………………………………………………………………… 3 1.2.1 The benefits of new enzymes 1.2.2 Limits of established methods for enzyme development 1.3 Evolving enzymes in vitro………………………………………………..… 6 1.3.1 Benefits of in vitro evolution 1.3.2 General workflow for in vitro methods 1.3.3 Library construction 1.4 Methods for in vitro directed evolution ………………………………….… 11 1.4.1 Ribosome display 1.4.2 mRNA display 1.4.3 In vitro compartmentalization (IVC) 1.4.4 DNA display 1.4.5 General principles and comparisons of different in vitro methods 1.5 General considerations for constructing artificial enzymes………………… 20 1.5.1 Identifying a target scaffold 1.5.2 Manipulating the protein scaffold 1.5.3 Testing and refinement of candidates 1.6 Rational design of artificial enzymes……………………………………….. 22 1.7 De novo selection of artificial enzymes…………………………………….. 24 1.8 Outlook for de novo selection………………………………………………. 27 Chapter 2: Highly Diverse Protein Library Based on the Ubiquitous (β/α)8 Enzyme Fold Yields Well-Structured Proteins Through In Vitro Folding Selection………….…………….. 30 2.1 Overview………………………………………………………………......... 30 2.2 Introduction……………………………………………………………….. 31 2.3 Results…………………………………………………………………….. 34 2.3.1 Identification and characterization of a (β/α)8 scaffold protein and an unfolded control 2.3.2 Optimization of the folding selection by in vitro protease digestion 2.3.3 Construction of intermediate libraries with randomized loops 2.3.4 Folding selections of the intermediate libraries by in vitro protease digestion v 2.3.5 Assembly of the final folding-enriched library 2.3.6 Analysis of stability of folding-enriched library and comparison to control library using the protease assay (in vitro) 2.3.7 Assessment of folding of the final libraries via GFP-fused reporter assay (in vivo) 2.3.8 Isolation of well-folded members of the final libraries by cell sorting 2.3.9 Analysis of soluble library-GFP fusions by Western blotting and SDS- PAGE 2.3.10 Biophysical characterization of soluble library clones 2.3.11 Sequence analysis of library clones 2.4 Discussion…………………………………………………………………. 47 2.5 Conclusion…………………………………………………………............ 51 2.6 Materials and methods……………………………………………….......... 52 2.6.1 Cloning and expression of GDPDwt and GDPDmut constructs: 2.6.2 Circular Dichroism (CD) spectroscopy: 2.6.3 1-Anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements: 2.6.4 Size exclusion chromatography: 2.6.5 Library assembly: 2.6.6 mRNA display: 2.6.7 In vitro folding selection by protease digestion: 2.6.7 GFP-based folding assay: 2.6.8 Western blot analysis: 2.7 Supplementary Information………………………………………………... 59 2.7.1 DNA sequence of the GDPDwt scaffold used as template for library assembly 2.7.2 Sequence alignment of the six soluble F(s) clones characterized in this chapter. Chapter 3: Thermostable artificial enzyme isolated by in vitro selection…………………………….….……………………… 70 3.1 Overview…………………………………………………………………… 70 3.2 Introduction……………………………………………………………….. 71 3.3 Results…………………………………………………………………….. 73 3.3.1 Setup of selection procedure 3.3.2 In vitro selection at 65 °C 3.3.3 Sequence analysis and expression of selected ligases 3.3.4 Activity of ligase enzymes 3.3.5 Characterization of thermal stability by Circular Dichroism (CD) 3.4 Discussion………………………………………………………………… 79 3.5 Conclusions………………………………………………………………. 83 3.6 Materials and methods……………………………………………………. 84 3.6.1 Preparation of oligonucleotides 3.6.2 Selection of RNA ligases at 65 °C vi 3.6.3 Expression & purification of RNA ligases 3.6.4 Screening for Ligase Activity by Gel-Shift assay 3.6.5 Determination of observed rate constants (kobs) 3.6.6 Circular Dichroism and thermal denaturation 3.7 Supporting information…………………………………………………… 87 3.7.1 Sequences of ligases selected at 65 °C 3.7.2 Oligonucleotide sequences
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