Expression and Purification of Aquaporin-6 in Different Systems Comparison of cell-free, Semliki Forest virus, and Pichia pastoris expression systems Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von André Krüger aus Hattingen, Deutschland Basel, 2012 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Andreas Engel und Prof. Dr. Henning Stahlberg Basel, den 26.06.2012 Prof. Dr. Martin Spiess Dekan Table of Contents 1. Introduction ........................................................................................................... 1 1.1 Biological memBranes: composition and function ........................................ 1 1.2 ‘Transport’ of water across biological memBranes ....................................... 1 1.1.1 Types and functions of human AQPs ........................................................................ 2 1.1.2 Aquaporin-6 ......................................................................................................................... 9 1.2 Heterologous expression of Aquaporins ........................................................ 18 1.2.1 Cell-free membrane protein expression .............................................................. 21 1.2.2 The Semliki Forest Virus expression system ...................................................... 26 1.2.3 The Pichia pastoris expression system ................................................................. 30 1.3 Aim of this work ..................................................................................................... 32 2 Methods ................................................................................................................ 35 2.1 Cell-free protein expression ............................................................................... 35 2.1.1 DNA template design .................................................................................................... 35 2.1.2 Transcription and translation ................................................................................... 35 2.1.2.1 Preparation of T7 RNA polymerase for cell-free expression .................................. 35 2.1.2.2 Preparation of E. coli S30 extract ........................................................................................ 36 2.1.2.3 Analytical and preparative scale cell-free expression ............................................... 37 2.1.2.4 Liposome preparation ............................................................................................................. 39 2.1.3 Purification ........................................................................................................................ 40 2.2 Protein expression with the Semliki Forest virus system ........................ 41 2.2.1 DNA template design .................................................................................................... 41 2.2.2 Transcription and translation ................................................................................... 41 2.2.2.1 In vitro transcription ................................................................................................................ 41 2.2.2.2 Transfection of mammalian cells ........................................................................................ 42 2.2.2.3 Harvesting and activation of recombinant virus ......................................................... 42 2.2.2.4 Titer determination .................................................................................................................. 43 2.2.2.5 Infection of mammalian cells and protein expression .............................................. 43 2.2.3 Purification ........................................................................................................................ 43 2.2.3.1 Membrane preparation and solubilization .................................................................... 43 2.2.3.2 Ni-NTA purification (IMAC) .................................................................................................. 44 2.2.3.3 Reconstitution ............................................................................................................................. 45 2.2.4 General cell culture methods .................................................................................... 45 2.3 Protein expression with Pichia pastoris ......................................................... 47 2.3.1 DNA template design .................................................................................................... 47 2.3.2 Transcription and translation ................................................................................... 48 2.3.3 Purification ........................................................................................................................ 48 2.4 Protein analysis ...................................................................................................... 50 2.4.1 Determination of protein concentration .............................................................. 50 2.4.2 SDS-PAGE ........................................................................................................................... 50 2.4.3 Western blot analysis ................................................................................................... 50 2.4.4 Size exclusion chromatography ............................................................................... 50 I EXPRESSION OF AQUAPORIN-6 IN DIFFERENT SYSTEMS 2.4.5 Single particle negative stain transmission electron microscopy ............ 51 2.5 Protein reconstitution into liposomes ............................................................ 52 2.6 Freeze fracture electron microscopy ............................................................... 54 2.6.1 Sample preparation ....................................................................................................... 54 2.6.2 Sample analysis ............................................................................................................... 54 2.7 Water conductance measurements of proteoliposomes ........................... 55 3 Results & Discussion ......................................................................................... 57 3.1 Cell-free expression of AQP6 .............................................................................. 57 3.1.1 Template design ............................................................................................................. 57 3.1.2 Transcription and translation .................................................................................. 58 3.1.2.1 Basic protocol ............................................................................................................................. 58 3.1.2.2 Buffer conditions ....................................................................................................................... 59 3.1.2.3 Optimizing parameters for expression ............................................................................ 59 3.1.2.4 Altering N-terminal tags to optimize transcription and translation ................... 61 3.1.2.5 Evaluation of detergents for expression and solubilization ................................... 62 3.1.3 Purification ....................................................................................................................... 65 3.1.4 Single particle analysis ................................................................................................ 69 3.1.5 Reconstitution into liposomes .................................................................................. 69 3.1.5 Co-translational reconstitution ................................................................................ 73 3.1.6 Water conductance of cell-free expressed AQP6 ............................................. 77 3.2 Semliki Forest virus expression of AQP6 ........................................................ 80 3.2.1 Template design ............................................................................................................. 80 3.2.2 Transcription ........................................................................................................................ 80 3.2.2.1 In vitro transcription .................................................................................................................. 80 3.2.2.2 Virus production ........................................................................................................................ 82 3.2.2 Translation ........................................................................................................................ 82 3.2.3 Solubilization ................................................................................................................... 84 3.2.4 Purification ....................................................................................................................... 85 3.2.5 Single particle analysis ................................................................................................ 89 3.2.6 Reconstitution into liposomes .................................................................................. 90 3.2.7 Water conductance of heterogously expressed AQP6 ................................... 93 3.3 Pichia pastoris expression of AQP6 .................................................................