HIGH-THROUGHPUT ASSAYS FOR BIOTIN PROTEIN LIGASE : A NOVEL ANTIBIOTIC TARGET by Belinda Ng Ling Nah, BSc in Industrial Biology (Hons) A thesis submitted to the University of Adelaide in fulfillment of the requirement for the degree of Master of Science School of Molecular & Biomedical Science The University of Adelaide Adelaide, South Australia, 5005 January 2009 TABLE OF CONTENTS TABLE OF CONTENTS……………………………………………………………………………………..i ABSTRACT…………………………………………………………………………………………………..iv STATEMENT OF ORIGINALITY…………………………………………………………………....…...vii ACKNOWLEDGEMENTS……………………………………………………………………………...…viii LIST OF FIGURES…………………………………………………………………………………………..ix LIST OF TABLES…………………………………………………………………………………………….x LIST OF PUBLICATIONS………………………………………………………………………………….xi LIST OF ABBREVIATION………………………………………………………………………………...xii CHAPTER 1 INTRODUCTION.................................................................................................. 1 1.1 NEEDS FOR NEW ANTIBIOTICS.................................................................................................. 1 1.2 BIOTIN....................................................................................................................................... 2 1.3 BIOTIN-DEPENDENT ENZYMES.................................................................................................. 3 1.4 BIOTIN DOMAINS....................................................................................................................... 4 1.5 BIOTIN PROTEIN LIGASE (BPL) .................................................................................................. 6 1.6 MICROBIAL BPLS....................................................................................................................... 6 1.6.1 Escherichia coli BPL (BirA)........................................................................................................7 1.6.2 Pyrococcus horikoshii BPL.......................................................................................................... 9 1.7 EUKARYOTIC BPLS.................................................................................................................. 10 1.8 AIMS AND SIGNIFICANCE ........................................................................................................ 11 1.8.1 Project Aims...............................................................................................................................11 1.8.2 Project Significance....................................................................................................................12 CHAPTER 2 GENERAL MATERIALS AND METHODS.................................................... 13 2.1 MATERIALS ............................................................................................................................ 13 2.1.1 General Materials .......................................................................................................................13 2.1.2 Chemical Reagents.....................................................................................................................14 2.1.3 Bacterial Strains .........................................................................................................................15 2.1.4 Bacterial Media ..........................................................................................................................15 2.1.5 Commercial Kits ........................................................................................................................15 2.1.6 Buffers and Solutions .................................................................................................................16 2.1.7 Radiochemical............................................................................................................................17 2.1.8 Peptide........................................................................................................................................17 2.1.9 Primers .......................................................................................................................................18 2.1.10 Plasmid.......................................................................................................................................19 2.1.11 Computer Software ....................................................................................................................19 2.1.12 Web Resources...........................................................................................................................19 i 2.2 GENERAL METHODS................................................................................................................ 20 2.2.1 Protein Techniques.....................................................................................................................20 2.2.1.1 Preparation of cell lysates...............................................................................................20 2.2.1.2 Determination of protein concentration ..........................................................................20 2.2.1.3 SDS-PAGE electrophoresis and gel staining...................................................................21 2.2.1.4 Western blot analysis.......................................................................................................21 2.2.1.5 MALDI mass spectrometry ..............................................................................................21 2.2.2 Molecular Biology Techniques ..................................................................................................22 2.2.2.1 Agarose gel electrophoresis ............................................................................................22 2.2.2.2 Preperation of DH5-! competent cells............................................................................ 22 2.2.2.3 Preparation of chemically competent E. coli...................................................................23 2.2.2.4 Restriction Digest of DNA ...............................................................................................23 2.2.2.5 Ligation of DNA fragments..............................................................................................24 2.2.2.6 Transformation of competent cells ..................................................................................24 2.2.2.7 Glycerol stocks ................................................................................................................24 2.2.2.8 Purification of Plasmid DNA...........................................................................................24 2.2.2.9 Quantification of DNA.....................................................................................................25 2.2.2.10 DNA sequencing ..............................................................................................................25 2.2.2.11 PCR Protocols.................................................................................................................25 2.2.3 In-vitro 3H-biotin biotinylation assay.........................................................................................26 CHAPTER 3 DEVELOPMENT AND CHARACTERIZATION OF A HIGH- THROUGHPUT ASSAY FOR E. COLI BPL ................................................... 27 3.1 INTRODUCTION ....................................................................................................................... 27 3.2 SPECIFIC METHODS ................................................................................................................. 29 3.2.1 Expression and purification of protein .......................................................................................29 3.2.1.1 E. coli BPL (BirA) ...........................................................................................................29 3.2.1.2 E. coli BCCP ...................................................................................................................31 3.2.2 BirA assay and fluorescence polarization ..................................................................................32 3.2.3 Peptide methods .........................................................................................................................33 3.2.4 Virtual screening for ATP analogues .........................................................................................33 3.2.5 Disc diffusion assay ...................................................................................................................34 3.2.6 Minimal inhibitory concentration (MIC) assay ..........................................................................34 3.3 RESULTS AND DISCUSSION...................................................................................................... 35 3.3.1 Principle of the assay..................................................................................................................35 3.3.2 Peptide 85-11 is a suitable BirA substrate..................................................................................36 3.3.3 Development of the assay...........................................................................................................37 3.3.4 Kinetic analysis of BirA.............................................................................................................38 3.3.5 Inhibition studies........................................................................................................................39 3.3.6