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Chapter 1 Introduction The Conditional Protein Splicing of Alpha-Sarcin: A model for inducible assembly of protein toxins in vivo. by Spencer C. Alford B.Sc., University of Victoria, 2004 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biochemistry and Microbiology Spencer C. Alford, 2007 University of Victoria All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author. ii Supervisory Committee The Conditional Protein Splicing of Alpha-Sarcin: A model for inducible assembly of protein toxins in vivo. by Spencer C. Alford B.Sc, University of Victoria, 2004 Supervisory Committee Dr. Perry Howard, Supervisor (Department of Biochemistry and Microbiology) Dr. Juan Ausio, Departmental Member (Department of Biochemistry and Microbiology) Dr. Robert Chow, Outside Member (Department of Biology) iii Supervisory Committee Dr. Perry Howard, Supervisor (Department of Biochemistry and Microbiology) Dr. Juan Ausio, Departmental Member (Department of Biochemistry and Microbiology) Dr. Robert Chow, Outside Member (Department of Biology) Abstract Conditional protein splicing (CPS) is an intein-mediated post-translational modification. Inteins are intervening protein elements that autocatalytically excise themselves from precursor proteins to ligate flanking protein sequences, called exteins, with a native peptide bond. Artificially split inteins can mediate the same process by splicing proteins in trans, when intermolecular reconstitution of split intein fragments occurs. An established CPS model utilizes an artificially split Saccharomyces cerevisiae intein, called VMA. In this model, VMA intein fragments are fused to the heterodimerization domains, FKBP and FRB, which selectively form a complex with the immunosuppressive drug, rapamycin. Treatment with rapamycin, therefore, heterodimerizes FKBP and FRB, and triggers trans-splicing activity by proximity association of intein fragments. Here, we engineered a CPS model to assemble inert fragments of the potent fungal ribotoxin, alpha (α)-sarcin, in vivo. Using this model, we demonstrate rapamycin-dependent protein splicing of α-sarcin fragments and a corresponding induction of cytotoxicity in HeLa cells. We further show that permissive extein context and incubation temperature are critical factors regulating the splicing of active target proteins. Ultimately, this technology could have potential applications in the fields of developmental biology and anti-tumour therapy. iv Table of Contents Supervisory Committee ...................................................................................................... ii Abstract..............................................................................................................................iii Table of Contents...............................................................................................................iv List of Tables ..................................................................................................................... vi List of Figures................................................................................................................... vii Abbreviations..................................................................................................................... ix Chapter 1 Introduction................................................................................................. 1 1.1 Overview and Research Premise ........................................................................ 1 1.2 The Engineering of Artificial Molecular Switches............................................. 6 1.2.1 Direct control of protein function ............................................................... 8 1.2.2 Chemical inducers of dimerization ............................................................. 8 1.3 Protein Splicing and Inteins.............................................................................. 11 1.3.1 Inteins and Genetic Mobility .................................................................... 11 1.3.2 Intein Discovery and Nomenclature ......................................................... 12 1.3.3 The Intein Splicing Mechanism................................................................ 15 1.3.3.1 Step 1: N-S/O Acyl Rearrangement...................................................... 16 1.3.3.2 Step 2: Transesterification .................................................................... 16 1.3.3.3 Step 3: Asparagine Cyclization and Branch Resolution....................... 17 1.3.3.4 Step 4: O/S-N Acyl Rearrangement...................................................... 17 1.3.4 Types of inteins......................................................................................... 18 1.3.4.1 Maxi-inteins ......................................................................................... 18 1.3.4.2 Mini-inteins.......................................................................................... 18 1.3.4.3 Alanine Inteins..................................................................................... 19 1.3.4.4 Trans-splicing Inteins .......................................................................... 19 1.3.5 Applications of Inteins.............................................................................. 21 1.4 Catalytic Protein Toxins: Inhibitors of Translation .......................................... 25 1.4.1 Alpha-Sarcin ............................................................................................. 27 1.4.2 Ricin.......................................................................................................... 29 1.5 Research Objectives.......................................................................................... 33 Chapter 2 Exogenous Expression of Protein Toxins in Mammalian Cells ................ 49 2.1 Introduction....................................................................................................... 49 2.2 Materials and Methods...................................................................................... 51 2.3 Results............................................................................................................... 60 2.3.1 Exogenous expression of α-sarcin and ricin in HeLa cells ...................... 60 2.3.2 Exogenous expression of Ricinus communis agglutinin A chain in HeLa cells ........................................................................................................................... 63 2.3.3 Characterization of α-sarcin and ricin mutants......................................... 64 2.4 Discussion......................................................................................................... 69 Chapter 3 Development and Characterization of an in vivo Conditional Protein Splicing Model for Protein Toxins. .......................................................... 94 3.1 Introduction....................................................................................................... 94 3.2 Materials and Methods...................................................................................... 95 3.3 Results............................................................................................................. 101 v 3.3.1 Establishment of conditional protein splicing (CPS) in mammalian cells…………………..…………………………………………………101 3.3.2 Development and characterization of a green fluorescent protein (GFP) CPS model in mammalian cells. ............................................................. 102 3.3.3 Development and characterization of an α-sarcin CPS model in mammalian cells…………… ................................................................. 104 3.3.4 Dose-dependency and temporal induction of α-sarcin splicing ............. 109 3.3.5 Comparison of promoter-driven expression and CPS-mediated expression of α-sarcin............................................................................................... 110 3.3.6 Splicing of α-sarcin in vivo decreases cell viability ............................... 111 3.3.7 Development and characterization of a ricin toxin A (RTA) CPS model in mammalian cells ..................................................................................... 112 3.4 Discussion....................................................................................................... 114 Chapter 4 Conclusions and Future Research .......................................................... 148 4.1 Conclusions..................................................................................................... 148 4.2 Future Research .............................................................................................. 149 4.2.1 Investigation into the cytotoxic mechanisms of α-sarcin and ricin........ 149 4.2.2 Optimizing conditional protein splicing models for protein toxins........ 151 4.2.3 Applications of a conditional protein splicing model for protein toxins 153 References....................................................................................................................... 158 vi List of Tables Table 2.1 Nomenclature and properties of mammalian toxin expression plasmids. ...... 75 Table 2.2 Oligonucleotide primer sequences used to generate α-sarcin, RTA, and RCA cDNAs....................................................................................................................... 76 Table 2.3 Overlap extension PCR cloning strategy
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