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Biochemical Analysis of a Prokaryotic Deubiquitinase from Escherichia Coli Cameron Wade Purdue University

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Biochemical Analysis of a Prokaryotic Deubiquitinase from Escherichia Coli Cameron Wade Purdue University Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations January 2016 Biochemical Analysis of a Prokaryotic Deubiquitinase from Escherichia Coli Cameron Wade Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Recommended Citation Wade, Cameron, "Biochemical Analysis of a Prokaryotic Deubiquitinase from Escherichia Coli" (2016). Open Access Theses. 1233. https://docs.lib.purdue.edu/open_access_theses/1233 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. *UDGXDWH6FKRRO)RUP 8SGDWHG PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance 7KLVLVWRFHUWLI\WKDWWKHWKHVLVGLVVHUWDWLRQSUHSDUHG %\ Cameron Wade (QWLWOHG BIOCHEMICAL ANALYSIS OF A PROKARYOTIC DEUBIQUITINASE FROM ESCHERICHIA COLI Master of Science )RUWKHGHJUHHRI ,VDSSURYHGE\WKHILQDOH[DPLQLQJFRPPLWWHH Chittaranjan Das Jean-Christophe Rochet Andrew D. Mesecar Mark C. Hall To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Chittaranjan Das $SSURYHGE\0DMRU3URIHVVRU V BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB $SSURYHGE\Timothy Zwier 04/21/2016 +HDGRIWKH'HSDUWPHQW*UDGXDWH3URJUDP 'DWH BIOCHEMICAL ANALYSIS OF A PROKARYOTIC DEUBIQUITINASE FROM ESCHERICHIA COLI A Thesis Submitted to the Faculty of Purdue University by Cameron Wade In Partial Fulfillment of the Requirements of the Degree of Master of Science May 2016 Purdue University West Lafayette, Indiana ii TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................. iv LIST OF FIGURES .............................................................................................................v LIST OF ABBREVIATIONS ........................................................................................... vii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION .....................................................................................1 1.1 Ubiquitination ......................................................................................................1 1.2 Deubiquitination ..................................................................................................4 1.3 Escherichia coli ...................................................................................................6 1.4 Bacterial Deubiquitinases ....................................................................................8 1.4.1 ElaD ..........................................................................................................10 CHAPTER 2 MATERIALS AND METHODS .............................................................13 2.1 Creation of Genetic Constructs ..........................................................................13 2.1.1 Cloning ......................................................................................................13 2.1.2 Mutagenesis ..............................................................................................14 2.1.3 Loop Swap From SdeA Inserted into ElaD wt .........................................15 2.2 Expression and Solubility ..................................................................................16 2.3 Affinity Purification ...........................................................................................17 2.3.1 GST Tagged Protein Purification..............................................................18 2.3.1.1 Purification after removal of GST Tag ............................................19 2.3.2 Ubiquitin Purification ...............................................................................20 2.4 Activity Assays ..................................................................................................23 2.4.1 Ubiquitin-Propargylamide Shift Assay .....................................................23 2.4.2 Diubiquitin Cleavage Assay .....................................................................23 2.4.3 Polyubiquitinated Green Fluorescent Protein Cleavage Assay ................24 2.4.4 Maltose Binding Protein-Nedd8-Ubiquitin Complex Cleavage Assay ....25 iii Page CHAPTER 3 RESULTS ................................................................................................26 3.1 Preparation of Protein ........................................................................................26 3.1.1 Cloning ......................................................................................................26 3.1.2 Mutagenesis ..............................................................................................28 3.1.3 Protein Expression and Solubility Test Small Scale .................................29 3.1.4 Protein Purification ...................................................................................31 3.2 Assays ................................................................................................................32 3.2.1 Ubiquitin-Propargylamide Shift Assay .....................................................32 3.2.2 Diubiquitin Cleavage Assay .....................................................................33 3.2.3 Polyubiquitinated Green Fluorescent Protein Cleavage Assay ................35 3.2.4 Maltose Binding Protein-Nedd8-Ubiquitin Complex Cleavage Assay ....38 CHAPTER 4 DISCUSSION ..........................................................................................40 4.1 ElaD Chain Linkage Preference .........................................................................40 4.2 ElaD Loop Interactions on Substrate Selectivity ...............................................41 4.3 Conclusion .........................................................................................................42 LIST OF REFERENCES ...................................................................................................44 APPENDICES Appendix A ..................................................................................................................48 Appendix B ..................................................................................................................50 Appendix C ..................................................................................................................52 iv LIST OF TABLES Tables Page 1.1 Strain Dependence of ElaD .................................................................................... 10 3.1 List of ElaD Constructs .......................................................................................... 29 v LIST OF FIGURES Figure Page 1.1 Mechanism of Ubiquitination ................................................................................... 2 1.2 Ubiquitination Chain Types ..................................................................................... 3 1.3 Structure of Ubiquitin Like Proteins ........................................................................ 4 1.4 Infection Cycle of Enteropathogenic and Enterohemorrhagic Escherichia coli ............................................................................................................................ 7 1.5 Eukaryotic Cell Autophagy Defense Mechanism .................................................... 8 1.6 Crystal Structure of SdeA and Ubiquitin .................................................................. 9 1.7 Model of ElaD and Ubiquitin ................................................................................. 11 1.8 Model of ElaD Superimposed on SdeA ................................................................. 12 2.1 Polymerase Chain Reaction Protocol ..................................................................... 15 2.2 Expression and Solubility Test for GST-ElaD wt .................................................. 17 2.3 GST-ElaD C313A GST Purification ...................................................................... 19 2.4 S75 Size Exclusion Chromatogram for ElaD C313A ............................................ 20 2.5 Ubiquitin Purification Using Ion Exchange ........................................................... 22 3.1 Agarose Gel of Cloning ElaD from Rosetta Cell ................................................... 27 3.2 Agarose Gel of Cloning ElaD L6 Chimera ............................................................ 28 3.3 Expression and Solubility Test of ElaD C313A and ElaD L6 ............................... 30 3.4 Expression and Solubility Test of ElaD Loop Point Mutants ................................ 31 3.5 Ubiquitin-Propargylamide Shift Assay for ElaD Constructs ................................. 33 vi Figure Page 3.6 ElaD wt Diubiquitin Cleavage Assay ..................................................................... 34 3.7 ElaD L6 Diubiquitin Cleavage Assay .................................................................... 34 3.8 K63 Q40A Diubiquitin Cleavage Assay ................................................................ 35 3.9 ElaD constructs GFP-Ubn Cleavage Assay ............................................................ 36 3.10 ElaD L6 Varied Concentration and Time GFP-Ubn
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