ABSTRACT CRUZ-TERAN, CARLOS ALBERTO. Engineering the Sso7d Scaffold for Biosensing Applications. (Under the direction of Balaji M. Rao). Current biosensor limitations include low thermal stability, complex protein immobilization protocols, and high cost. In this thesis, we show how the hyperthermophilic protein scaffold Sso7d can be modified by protein engineering to address some of these limitations. While non-specific adsorption is widely used for immobilizing proteins on surfaces, proteins may become immobilized with occluded active sites due to the random nature of this process. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. We generated a combinatorial protein library by randomizing six residues in a binding protein derived from the Sso7d scaffold; mutations were targeted in a region far from the binding site. This library was screened to isolate binders that retain binding to its cognate target as well as exhibit adsorption on silica. A single mutant – Sso7d-2B5 – was characterized. We demonstrated that silica beads coated with Sso7d-2B5 could achieve up to seven-fold higher capture of target than beads coated with the parent protein. Thus, we provide a generalizable approach for introducing mutations in proteins to improve their activity upon immobilization. Magnetization of yeast cells is important for the generation of biocatalysts, biosorbents, and yeast recovery. For this process to work yeast cells must be able to become magnetized in complex mixtures. We hypothesized that we could isolate proteins to anchor yeast on iron(II,III) oxide, thus rendering the cells magnetic. A yeast surface display library of Sso7d mutants was panned against iron oxide under stringent conditions. After six rounds of selection, a single Sso7d mutant (SsoFe2) was characterized. Yeast cells expressing SsoFe2 could be magnetized in complex mixtures where magnetization of yeast cells not expressing SsoFe2 was poor. Adding titanium(IV) or silicon dioxides did not decrease SsoFe2-yeast magnetization, suggesting that SsoFe2 is specific to iron oxide. Overall, we demonstrate the usefulness of yeast surface display for isolating proteins with strong binding for a material’s surface. Mix-and-read assays are useful for detecting analytes in complex biological fluids without the need for washes. An ideal assay should produce a fast response and have a high signal to noise ratio. We previously designed a mix-and-read assay for lysozyme detection based on tripartite GFP (green fluorescent protein) reconstitution. However, 4 hours were required for reliable lysozyme detection. We sought to increase the response time of this system by replacing split-GFP with fragments of split-NanoLuc luciferase. Indeed, lysozyme could be detected immediately after adding luciferase substrate, without loss of sensitivity. These results demonstrate that the subunits of bivalent Sso7d binders isolated from combinatorial pairwise assembled libraries can be adapted to different split-protein complementation systems. Simultaneous surface display and secretion of proteins would increase the speed at which mutants isolated from combinatorial libraries can be characterized. To this end, we developed a system in Saccharomyces cerevisiae for simultaneous surface display and secretion of proteins based on ribosomal skipping. A “self-cleaving peptide” with 50% skipping efficiency (F2A) was placed in between the protein to be displayed and the anchor protein Aga2, downstream of a secretion leader. Consequently, half of the translation product corresponds to protein-Aga2 fusions, which get displayed on the yeast surface, while the other half are proteins with a secretion tag. Secretion and display of two Sso7d mutants and glucose oxidase from Aspergillus Niger is demonstrated. We also show secretion and display of glucose oxidase and Sso7d in C-terminal configuration, where Aga2 is placed downstream of a secretion signal followed by F2A and the protein of interest. Thus, we present a versatile secretion and display system in Saccharomyces cerevisiae. © Copyright 2015 Carlos Alberto Cruz-Teran All Rights Reserved Engineering the Sso7d Scaffold for Biosensing Applications by Carlos Alberto Cruz-Teran A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical and Biomolecular Engineering Raleigh, North Carolina 2017 APPROVED BY: _______________________________ _______________________________ Balaji M. Rao Jason M. Haugh Committee Chair Minor Member _______________________________ _______________________________ Jan Genzer Troy Ghashghaei DEDICATION I would like to dedicate this thesis to my grandpa, who week after week took interest in my progress until his last days, and to my family, who has always been there to support me. Para mi abuelo, que semana a semana estuvo pendiente del progreso en mi investigación, y para mi familia por siempre estar ahí para apoyarme. ii BIOGRAPHY Carlos Alberto Cruz-Teran was born in Quito, Ecuador, in 1989. From a young age, Carlos was interested in science. After finishing high school, Carlos started his undergraduate studies in Chemical Engineering at the University of New Mexico, Albuquerque. It was at UNM where Carlos finally became part of a research team. Dr. Claudia Lurhs kindly allowed Carlos to help her investigate the fabrication of hollow tungsten oxide nanotubes. This increased Carlos’ interest in nanomaterials, motivating him to pursue more research opportunities in this field. Carlos joined Dr. Marek Osinski’s lab at the Center of High Technology Materials, UNM, to investigate the fabrication of nanoparticles for “water splitting” applications. However, Carlos’ undergraduate adviser, Dr. Eva Chi, stirred Carlos’ interest into bioengineering related research, particularly protein engineering. Consequently, Carlos joined Dr. Chi’s lab to study a novel method for encapsulation of proteins in silica hydrogels. This experience highly increased Carlos’ interests in protein engineering. After graduating from UNM in 2011, Carlos started a PhD program in Chemical Engineering at North Carolina State University, Raleigh. In the fall of 2011, he joined Dr. Bala Rao’s protein engineering lab. Coincidentally, a significant portion of Carlos’ PhD research involved understanding protein-material interactions, thus bridging together his interests in nanomaterials and protein engineering. Carlos will start a postdoctoral position at the University of North Carolina, Chapel Hill after graduation. Carlos’ career goal is to join a research team in industry where he could contribute to the development of new protein therapeutics or drug delivery systems. iii ACKNOWLEDGMENTS I would like to thank my adviser Dr. Rao, for all the years of funding, support, and guidance he has provided me through my PhD career. Thank you for helping me become a better researcher and encouraging me to be curious and always give the best of myself. I would like to thank Dr. Nimish Gera, from whom I learned a lot about protein engineering and research when I first joined the lab, and who has been an invaluable resource even after graduating from the lab. Thank you, Dr. Mahmud Hussain, and Dr. Prasenjit Sarkar, for all your help as well. I would also like to thank Adam Mischler, Dr. Kevin Carlin, and Dr. Karthik Tiruthani for always being ready to help me and discuss about science and stuff when we were labmates. I would also like to thank you for making all those years in the lab more enjoyable. I am very grateful for all the time Dr. Kirill Efimenko spent training me in ellipsometry and helping me characterize some of my samples by different techniques. Special thanks to Dr. Jan Genzer and Dr. Efimenko, whose guidance and comments were essential for publishing one of my manuscripts. I would like to thank Dr. Bob Kelly, who was always ready to give me advise and allowing me to use his lab equipment. I would like to recognize the hard work of all the undergraduate students I was lucky to work with, Rachel Snyder, Nick Capets, McKelvey Bump, Bhavana Kaki, Ula Watso, Stephen Ryan, Sara Knowlson, Apoorva Tharavarty, and Nikki McArthur. I hope I was a good mentor and that you enjoyed the time you spent in the lab. Special thanks to Apoorva Tharavarty and Nikki McArthur, with whom I have been lucky to work with for almost two years, and whose hard work and dedication have been invaluable for completing this thesis. iv At last but not least, I would like to thank my mom, dad, brother, and sister for always being there to support me throughout the way. Whether it has been through Skype, WhatsApp, or Facetime, I feel like you have been by my side throughout all these years. v TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................. xi CHAPTER 1 ............................................................................................................................. 1 Addressing biosensor limitations by engineering of the Sso7d scaffold .................................. 1 1.1 Introduction ....................................................................................................................