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Developing Methods to Understand and Engineer Protease Cleavage Specificity Developing methods to understand and engineer protease cleavage specificity A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Michael Douglas Lane IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Burckhard Seelig September 2016 © Michael D Lane, 2016 Acknowledgements I would like to thank the many people who supported me along the way towards completing my thesis. Above all, I thank my advisor, Dr. Burckhard Seelig, who gave me the opportunity to pursue my ideas, embraced my research with thoughtful criticism and relentless optimism, and shaped my scientific approach through challenging me to be the best I can be. I also would like to thank previous members of the Seelig lab, Dr. Misha Golynskiy, Dr. John Haugner, Dr. Dana Morrone, and Dr. Aleardo Morelli, for being good friends and the many scientific discussions we had that guided me through the rough experiments. To the new Seelig lab members, especially Dr. Matilda Newton, Dr. Yari Cabezas, and Dr. Nisha Kanwar, thank you for your critical feedback, friendship, and taking away all my lab responsibilities. To the MSTP, I would like to thank Dr. Yoji Shimizu, Susan Shurson, and Nicholas Berg for their unending help guiding through the woven MD/PhD program. In particular, I thank the MSTP leadership for allowing me to pursue the program tailored to my interests. To my thesis committee, thank you for your support through the years. From annual reviews, to informal discussions, to grant application drafts, your feedback molded my abilities and experience. To my friends and family, especially my wife, Dr. Kathleen Lane, thank you for keeping me going through tough times, and understanding when I disappeared for weeks or months at a time. I would like to acknowledge the NIH MSTP Training Grant (T32 GM008244), American Heart Association Predoctoral Fellowship, NIH Exploratory/Developmental Research Grant (R21 AI113406), and the Doctoral Dissertation Fellowship for funding my training and making my ideas a reality. Finally, I would like to thank the administration of the Biotechnology Institute and the Biochemistry, Molecular Biology, and Biophysics Department for creating a remarkable research environment and training program. And to all the researchers in the Gortner Laboratory, thank you for making this old bunker a place of inspiring thought and collaboration. i Dedication For my parents, Barry and Lyn Lane, who raised me to appreciate rational thought, cultivated my curiosity and creativity, and inspired me to push myself as hard as I can. ii Abstract Proteases are ubiquitous enzymes that comprise nearly 2% of all human genes. These robust enzymes are attractive potential therapeutics due to their catalytic turnover and capability for exquisite specificity. While most existing drugs require a stoichiometric ratio to function, therapeutic proteases could clear their targets much more efficiently. Unfortunately, existing technologies are inadequate for understanding and engineering therapeutic proteolytic specificities. My thesis work has focused on building the groundwork to enable these technologies to thrive. For the goal of engineering a new protease, it is currently necessary to identify prototype proteases for engineering efforts that have specificities similar to the desired target substrate. Current technologies are unable to characterize proteases adequately for this goal. Accordingly, I invested in developing a method for the accurate characterization of protease cleavage specificity. Our unique combination of mRNA display technology, Next-Generation Sequencing, and mass spectrometry enables the sampling of all possible permutations of octamer substrates and the identification of millions of cleavage sites. The throughput of our approach is orders of magnitude greater than the current state-of-the-art methods. The resulting high-resolution specificity maps can be applied to identify promising protease prototypes, predict human cross-reactivity, or lead to a better understanding of this critical component of natural physiology. In the work presented here, I applied my new specificity-screening method to assess the specificities of the proteases factor Xa, ADAM17, and streptopain. The resulting cleavage preference maps confirmed known specificities, and revealed new insight into the broad preferences of both narrow- and broad-specificity proteases. In particular, disfavored amino acids were illuminated better than ever before. The next focus of my work was to engineer multiple-subsite novel protease specificity. I chose streptopain as the prototype for my efforts to neutralize the superantigen exotoxin SpeA. I identified a target loop of SpeA wherein cleavage would result in inactive fragments. Further, I confirmed that streptopain can be successfully presented as an mRNA displayed fusion. iii In summary, my thesis work established crucial methodologies for applying mRNA display technology to enable the understanding and ultimately engineering the specificity of therapeutic proteases. iv Table of Contents Acknowledgements .............................................................................................................. i Dedication ........................................................................................................................... ii Abstract .............................................................................................................................. iii Table of Contents ................................................................................................................ v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1: Introduction ................................................................................ 1 1.1 Thesis overview ................................................................................................ 1 1.2 Significance....................................................................................................... 3 1.2.1 The untapped potential of proteases as therapeutics ......................................... 3 1.2.2 Protease specificity drives their unique function .............................................. 3 1.2.3 Limited technologies hamper progress towards therapeutic proteases ............. 4 1.2.4 Engineering therapeutic proteases can defend against bacterial exotoxins ...... 5 1.3 Current methods for characterizing protease specificity .................................. 7 1.3.1 Probing specificity with chemically synthesized peptide libraries ................... 7 1.3.2 Probing specificity with large combinatorial libraries ...................................... 8 1.3.3 Current methods are too limited to characterize promiscuous proteases ........ 11 1.4 Current methods for engineering protease specificity .................................... 14 1.4.1 Specificity engineering of protease OmpT by cell-surface display ................ 14 1.4.2 TEV protease specificity engineering by YESS ............................................. 15 1.4.3 Computational design of α-gliadin peptidase ................................................. 15 1.4.4 Limitations of current protease specificity engineering efforts ...................... 16 1.5 Advances in the directed evolution of proteins ............................................... 17 1.5.1 Overview ......................................................................................................... 17 1.5.2 Introduction ..................................................................................................... 17 1.5.3 Advancing selection technologies................................................................... 18 1.5.4 Expanding the scope of selections to new properties ..................................... 24 1.5.5 Conclusion ...................................................................................................... 28 1.6 Outlook ........................................................................................................... 29 Chapter 2: Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity ...........................................................................................................31 2.1 Overview ......................................................................................................... 31 2.2 Introduction ..................................................................................................... 32 2.3 Materials and methods .................................................................................... 34 2.3.1 Materials ......................................................................................................... 34 2.3.2 Overexpression of streptopain by autoinduction in E. coli ............................. 34 2.3.3 Purification of streptopain by affinity chromatography .................................. 35 2.3.4 Confirmation of streptopain protein identity by mass spectrometry .............. 35 v 2.3.5 Proteolytic activity measured by azocasein assay .......................................... 36 2.3.6 Assay of streptopain cleavage specificity using peptides substrates .............. 37 2.4 Results ............................................................................................................
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