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Expression of Human Α-N-Acetylglucosaminidase in Sf9 Insect Cells: Effect of Cryptic Splice Site Removal and Native Secretion-Signaling Peptide Addition

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Expression of Human Α-N-Acetylglucosaminidase in Sf9 Insect Cells: Effect of Cryptic Splice Site Removal and Native Secretion-Signaling Peptide Addition Expression of Human α-N-Acetylglucosaminidase in Sf9 Insect Cells: Effect of Cryptic Splice Site Removal and Native Secretion-Signaling Peptide Addition by Roni Rebecca Jantzen B.Sc., University of Victoria, 2009 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in the Department of Biology Roni Rebecca Jantzen, 2011 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 Expression of Human α-N-Acetylglucosaminidase in Sf9 Insect Cells: Effect of Cryptic Splice Site Removal and Native Secretion-Signaling Peptide Addition by Roni Rebecca Jantzen B.Sc., University of Victoria, 2009 Supervisory Committee Dr. Francis Y. M. Choy, Supervisor (Department of Biology) Dr. Robert L. Chow, Departmental Member (Department of Biology) Dr. C. Peter Constabel, Departmental Member (Department of Biology) iii Abstract Supervisory Committee Dr. Francis Y. M. Choy, Supervisor (Department of Biology) Dr. Robert L. Chow, Departmental Member (Department of Biology) Dr. C. Peter Constabel, Departmental Member (Department of Biology) Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. iv Table of Contents Supervisory Committee ...................................................................................................... ii Abstract..............................................................................................................................iii Table of Contents............................................................................................................... iv List of Tables ..................................................................................................................... vi List of Figures................................................................................................................... vii List of Abbreviations ......................................................................................................... ix Acknowledgments............................................................................................................. xii Dedication........................................................................................................................xiii 1. Introduction.................................................................................................................. 1 1.1 Mucopolysaccharidosis III Type B ........................................................................... 1 1.1.1 Overview of Mucopolysaccharidosis III.......................................................... 1 1.1.2 MPS IIIB Clinical Manifestations and Prognosis............................................ 1 1.1.3 Neurological Effects ........................................................................................ 3 1.1.4 Causative Mutations Identified ........................................................................ 6 1.1.5 Current and Proposed Therapies...................................................................... 8 1.1.6 Brief Physiology of the Blood-Brain Barrier................................................. 11 1.2 α-N-Acetylglucosaminidase and Heparan Sulphate ............................................... 12 1.2.1 Human α-N-Acetylglucosaminidase Gene and Enzyme Characteristics ...... 12 1.2.2 Heparan Sulphate Metabolism....................................................................... 13 1.2.3 Heparan Sulphate in the Nervous System...................................................... 15 1.3 Possibility of a Cryptic Splice Site in Naglu cDNA ............................................... 18 1.3.1 Cryptic Splice Site Overview......................................................................... 18 1.3.2 Evidence for Naglu mRNA Degradation Due to Cryptic Splicing................ 20 1.3.3 Removal of Proposed Cryptic Splice Sites by Site-Directed Mutagenesis.... 21 1.4 Expression of Human Recombinant Naglu ............................................................. 22 1.4.1 Spodoptera frugiperda Insect Cell System .................................................... 22 1.4.3 Vector pIZT/V5-His....................................................................................... 23 1.4.4 Purification of Naglu...................................................................................... 24 1.4.5 HIV-TAT–Derived Protein Transduction Domains ...................................... 25 1.5 Project Overview and Objectives ............................................................................ 28 2. Materials and Methods............................................................................................... 31 2.1 Materials.................................................................................................................. 31 2.1.1 Chemicals and Reagents ................................................................................ 31 2.1.2 Prepared Media and Solutions ....................................................................... 32 2.1.3 Equipment and Software................................................................................ 33 2.1.4 Cell Lines ....................................................................................................... 34 2.1.5 Materials Contributed by Others.................................................................... 34 2.2 Methods ................................................................................................................... 35 2.2.1 Creation of Gene Constructs.......................................................................... 35 2.2.1a Gene Constructs pIZT/Kozak-Naglu and pIZT/WT-Naglu.................... 35 2.2.1b Gene Constructs pIZT/NNSS-Naglu, pIZT/NNSS-Naglu-PTD4, pIZT/NNSS-ΔSNaglu, and pIZT/NNSS-ΔSNaglu-PTD4 ...................... 37 v 2.2.2 Cloning and Expression in E. Coli................................................................. 41 2.2.3 Transfection and Selection of Sf9 Insect Cells .............................................. 42 2.2.4 Sf9 Cell Growth and Expression.................................................................... 42 2.2.5 Bradford Protein Concentration, GFP Fluorescence, and Naglu Activity Assays ............................................................................................................ 46 2.2.6 RNA Extraction, RT-PCR, and Genomic DNA Analysis.............................. 47 2.2.7 Protein Purification ........................................................................................ 48 2.2.8 Protein Detection and Visualization .............................................................. 50 3. Results........................................................................................................................ 53 3.1 Creation, Cloning, and Sequencing of Naglu cDNA Constructs ............................ 53 3.1a Gene Constructs pIZT/Kozak-Naglu and pIZT/WT-Naglu........................... 53 3.1b Gene Constructs pIZT/NNSS-Naglu, pIZT/NNSS-Naglu-PTD4, pIZT/NNSS-ΔSNaglu, and pIZT/NNSS-ΔSNaglu-PTD4 ............................. 53 3.2 Transfection and Selection of Sf9 Insect Cells........................................................ 56 3.3 Overall Production Levels of Recombinant Naglu Protein..................................... 57 3.4 Naglu Expression in Sf9: Kozak Project................................................................. 62 3.5 Naglu Expression in Sf9: Cryptic Splice Site Project ............................................. 63 3.5a Transient Expression of Various Naglu Gene Constructs in Sf9 .................... 63 3.5b Stable Expression of Various Naglu Gene Constructs in Sf9 ......................... 68 3.6 Effectiveness of the Native Naglu Secretion Signal in Sf9..................................... 72 3.7 Purification Trial (Phenyl SepharoseTM Hydrophobic Interaction Column)........... 80 4. Discussion.................................................................................................................. 87 4.1 Transfection and Selection
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