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Expanding the Uses of Split-Intein Through Protein Engineering Expanding The Uses of Split-Intein Through Protein Engineering by Stanley Siu Cheung Wong A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Stanley Siu Cheung Wong 2013 Expanding the Uses of Split-Intein Through Protein Engineering Stanley Siu Cheung Wong Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2013 Abstract Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems ha ve been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. ii First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions. iii Acknowledgements My supervisor, Dr. Kevin Truong, for his constant encouragement, guidance, and invaluable ideas. My past and present lab friends and colleagues, for their friendship, technical assistance, and moral support. My supervisory committee, for their mentoring, insights, and challenging me. My friends and family for their support, patience and encouragement. iv Table of Contents Abstract........................................................................................................................................... ii Acknowledge ments ....................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Publications ....................................................................................................................... ix List of Abbreviations .....................................................................................................................x List of Figures................................................................................................................................ xi List of Movies .............................................................................................................................. xiii 1 Introduction............................................................................................................................ 1 1.1 Motivation ....................................................................................................................... 1 1.2 Research Objectives ....................................................................................................... 2 1.3 Organization ................................................................................................................... 2 2.1 Split-Protein Systems ..................................................................................................... 4 2.2 Inteins .............................................................................................................................. 7 2.2.1 Protein Splicing Pathway ....................................................................................... 7 2.2.2 Split Intein ............................................................................................................. 10 2.2.3 Naturally Split Intein ............................................................................................ 11 2.2.4 Controlling Protein Splicing – Optogenetics ...................................................... 13 2.3 Recurring Model Proteins ........................................................................................... 15 2.3.1 Genetically Encoded Ca2+ Indicato rs .................................................................. 16 2.3.2 Rho GTPases ......................................................................................................... 17 2.3.3 Caspase-7 ............................................................................................................... 18 3.1 Gene Construction – Subcloning ................................................................................ 19 3.1.1 Materials, Reagents, and Equipment .................................................................. 19 v 3.1.2 Generating a Cassette from a New Gene of Interest ......................................... 22 3.1.3 Generating Fusion Proteins from Two Cassettes............................................... 25 3.1.4 Removing Fluorescent Protein Gene................................................................... 28 3.1.5 Other Strategies for Improving Fluorescent Screening and Gene Insertion .. 29 3.2 In Vitro Pro tein Analysis – SDS-PAGE ..................................................................... 32 3.3 Cell Culture and Live-Cell Imaging ........................................................................... 34 3.3.1 Cell Culture Reagents, Materials, and Equipment ............................................ 34 3.3.2 Procedures for Cell Culture, Tra nsfectio n, a nd Sto rage ................................... 35 3.3.3 Live-Cell Imaging Equipment ............................................................................. 37 3.3.4 Imaging Procedures .............................................................................................. 38 3.4 Data Analysis ................................................................................................................ 39 3.4.1 Statistical Comparison Analysis .......................................................................... 39 3.4.2 Quantification of Co-Localization ....................................................................... 39 3.4.3 Generation, Imaging and Tracking of Transgenic C. Elegans ......................... 39 4 Reassembly of Split GECIs ................................................................................................. 41 4.1 Chapter Introduction and Aims.................................................................................. 41 4.2 Results ........................................................................................................................... 42 4.2.1 General Strategy of the Visual Assay .................................................................. 43 4.2.2 Establishing Protein Trans-Splicing Assay in Mammalian Cells ..................... 43 4.2.3 Reassembly of TN-XL Ca2+ Biosensor by NpuDnaE Protein Trans-Splicing . 48 4.2.4 Reassembling GCaMP2 Ca2+ Biosensor by NpuDnaE Protein Trans-Splicing50 4.2.5 Reassembly of GCaMP2 in Pharyngeal Muscle Cells of C. Elegans ................ 54 4.3 Discussion ...................................................................................................................... 56 4.4 Chapter Conclusions .................................................................................................... 57 5 Simultaneous Reassembly of Two Target Proteins .......................................................... 59 vi 5.1 Cha pter Introduction and Aims.................................................................................. 59 5.2 Results ........................................................................................................................... 60 5.2.1 General Strategy for Reassembling Two Target Proteins Using a Single NpuDnaE Intein ................................................................................................................... 61 5.2.2 NpuDnaE Inteins Reassembles Two Target Proteins ........................................ 62 5.2.3 NpuDnaE PTS Can Restore RhoA Activity and Venus Fluorescence ............. 66 5.2.4
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