Characterisation of a Putative Serine Protease Expressed in vivo by Mycobacterium avium subsp. paratuberculosis. Rona Mary Cameron Presented for the degree of PhD The University of Edinburgh 1996 a Table of Contents. Declaration .................................................................................................... Aknowledgements ..........................................................................................ii Abstract ......................................................................................................... Abbreviations ................................................................................................v Chapter 1 Introduction 1.1 The Mycobacterwceae ........................................................................1 1.1.1 The Cell Wall of Mycobacteria .............................................................1 1. 1.2 Mycobacterial Pathogens and Diseases .................................................. 5 1.2 The M. avium Complex (MAC) .......................................................... 6 1 .2.1 Members of the MAC ..........................................................................6 1.2.2 Molecular Characterisation of the MAC ................................................7 1 .2.3 Taxonomy of the MAC ........................................................................16 1.3 Known Genes and Proteins of M.a. avium, M.a. sil;'aticum and M.a. paratuberculosis ................................................................................... 19 1.4 Johne's Disease ...................................................................................24 1.4.1 Aetiological Agent .................................................................................24 1.4.2 Disease and Transmission ......................................................................25 1.4.3 Immunology of Johne's Disease ............................................................26 1.4.4 Diagnosis of Johne's Disease .................................................................28 1.5 Crohn's Disease ...................................................................................32 1.6 Identification of Genes and Proteins Expressed in vivo by M.a. paratuberculosis .................................................................................... 34 Chapter 2 Materials and Methods 2.1 Bacterial Strains and Plasmids .............................................................37 2.1.1 Escherichia coli ..................................................................................... 37 2.1.2 Mycobacteria .........................................................................................38 2.1.3 Plasmid Vectors .....................................................................................38 2.2 Chemicals and Culture .........................................................................39 2.3 Gel Electrophoresis of Proteins 39 2.3.1 SDS Polyacrylamide Gel Electrophoresis (SDS PAGE) ........................... 39 2.3.2 Native Polyacrylarnide Gel Electrophoresis ............................................. 40 2.3.3 Western Blotting and Immunoscreening .................................................. 40 2.3.4 Coomassie Blue Staining of SDS PAGE Gels ......................................... 41 2.3.5 Silver Staining of SDS PAGE Gels ......................................................... 41 2.4 Production of Recombinant Fusion Protein ......................................... 41 2.4.1 Culture and Induction ............................................................................. 41 2.4.2 Purification of 13-Galactosidase Fusion Proteins ....................................... 42 2.4.3 Factor Xa Cleavage of 3-Galactosidase Fusion Proteins ........................... 42 2.4.4 Purification of Glutathione S Transferase Fusion Proteins ........................ 42 2.4.5 Thrombin Cleavage of Glutathione S Transferase Fusion Proteins ............ 43 2.5 Purification of Recombinant S8 Protein ...............................................43 2.5.1 Culture and Induction .............................................................................43 2.5.2 Osmotic Shock of Escherichia coii ......................................................... 44 2.5.3 Chromatographic Purification of the S8 Recombinant Protein ..................44 2.5.4 Isoelectric Focusing ................................................................................ 45 2.6 Antibody Elution ..................................................................................45 2.7 Purification of Native Protein ............................................................... 46 2.7.1 Production of Ascitic Fluid ..................................................................... 46 2.7.2 Purification of IgGi from Ascitic Fluid .................................................... 46 2.7.3 Construction of Monoclonal Affinity Column .......................................... 47 2.7.4 Affinity Purification of Native Protein .................................................... 47 2.7.5 Protein Amino Terminal Sequence Analysis ............................................. 49 2.8 Serine Protease Assays .......................................................................... 50 2.8.1 Digestion of a and 3 Casein .................................................................... 50 2.8.2 Casein Gel Assay .................................................................................... 50 2.8.3 Agar Gel Diffusion Assays ...................................................................... 50 2.8.4 Digestion of Casein by Recombinant Escherichia coil .............................. 50 2.8.5 Sensitivity to Hydrogen Peroxide ............................................................. 51 2.9 Nucleic Acid Preparation and Manipulation .........................................51 2.9.1 Extraction of High Molecular Weight DNA from Mycobacteria ................51 2.9.2 Small Scale Plasmid Preparation from Escherichia coil ............................52 2.9.3 Large Scale Plasmid Preparation from Escherichia co/i ............................53 2.9.4 Preparation of Single Stranded DNA from Escherichia coil .....................54 2.9.5 Agarose Gel Electrophoresis ................................................................... 55 2.9.6 Purification of DNA Fragments from Agarose ......................................... 55 2.10 DNA Sequencing .................................................................................. 55 2.11 Polymerase Chain Reaction (PCR) .......................................................56 2.11.1 PCR of S8 DNA Insert ........................................................................... 56 2.11.2 PCR of 16S Ribosomal RNA Gene ......................................................... 57 2.11.3 Detection of PCR Products on Acrylamide Gels ....................................... 57 2.12 Cloning PCR Products ......................................................................... 58 2.12.1 Ligation into Vector DNA ....................................................................... 58 2.12.2 Preparation of Competent Cells ............................................................... 58 2.12.3 Transformation of Competent Cells ......................................................... 58 2.13 Nucleic Acid Hybridisation ...................................................................59 2.13.1 Southern Blotting ....................................................................................59 2.13.2 Dot Blotting DNA ..................................................................................59 2.13.3 Labelling DNA Probes by Random Priming .............................................60 2.13.4 Hybridisation and Autoradiography ..........................................................60 2.14 Site Directed Mutagenesis .....................................................................61 Chapter 3 Characterisation of Recombinant Clones 3.1 Introduction ..........................................................................................63 3.2 Results ..................................................................................................65 3.2.1 Expression and Purification of Fusion proteins .........................................65 3.2.2 Identification of Native Protein ................................................................73 3.2.3 Characterisation of the S4 and S8 DNA Inserts ........................................ 75 3.3 Discussion .............................................................................................79 Chapter 4 Secretion of S8 Recombinant Protein from Escherichia coli. 4.1 Introduction ..........................................................................................87 4.2 Results ..................................................................................................88 4.2.1 PCR of S8 Open Reading Frame .............................................................88 4.2.2 Cloning S8 PCR Product into pUC18 .....................................................90 4.2.3 Expression of Recombinant Protein from pUCMS/S8 ..............................91 4.2.4 Purification of Recombinant S8 by Anion Exchange Chromatography .......93 4.2.5 Purification of Recombinant S8 by Isoelectric Focusing ............................96 4.3 Discussion .............................................................................................101 Chapter 5 Expression of S8 recombinant Protein from the