Technische Universität München Fakultät für Maschinenwesen Lehrstuhl für Bioverfahrenstechnik Crystal Contact Engineering to Enhance Protein Crystallization Processes Phillip Grob Vollständiger Abdruck der von der Fakultät für Maschinenwesen der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation Vorsitzender: Prof. Wolfgang Polifke, Ph.D. Prüfer: 1. Prof. Dr.-Ing. Dirk Weuster-Botz 2. Prof. Dr. rer. nat. Michael Groll Die Dissertation wurde am 28.05.2020 bei der Technischen Universität München eingereicht und durch die Fakultät für Maschinenwesen am 20.10.2020 angenommen. OléOléBioVT Contents 1 Introduction ...................................................................................................................... 1 2 Motivation and Objective .............................................................................................. 3 3 Theoretical Background ................................................................................................ 7 3.1 Proteins ..................................................................................................................................... 7 3.1.1 Structures and Fundamental Interactions ...................................................... 7 3.1.2 Protein Engineering Strategies ....................................................................... 12 3.1.3 Biomanufacturing of Recombinant Proteins ............................................... 13 3.1.4 Lactobacillus brevis Alcohol Dehydrogenase (LbADH) ............................ 17 3.2 Protein Crystallization ........................................................................................................ 18 3.2.1 Fundamentals ..................................................................................................... 19 3.2.2 Crystallization Methods and Agents ............................................................. 23 3.2.3 Crystallography ................................................................................................. 25 3.2.4 Protein Crystal Engineering ........................................................................... 27 3.2.5 Technical Protein Crystallization .................................................................. 29 4 Material and Methods................................................................................................... 35 4.1 Material ................................................................................................................................... 35 4.2 Molecular Biological Methods ........................................................................................... 35 4.2.1 Site-Directed Mutagenesis ............................................................................... 35 4.2.2 DNA Separation by Agarose Gel Electrophoresis ..................................... 36 4.2.3 DpnI Digestion ................................................................................................... 36 4.2.4 Plasmid DNA Amplification, Extraction and Sequencing ....................... 36 4.2.5 Molecular Subcloning ....................................................................................... 36 4.3 Bacterial Transformation and Protein Production ....................................................... 37 4.3.1 Production of Chemically Competent Bacterial Cells .............................. 37 4.3.2 Heat Shock Transformation of Chemically Competent Cells ................. 38 4.3.3 Recombinant Protein Production with E. coli ............................................. 38 4.3.4 OD600 and Biomass Determination ................................................................ 40 4.3.5 Cell Harvest and Disruption ........................................................................... 40 4.4 Protein Purification and Processing ................................................................................ 41 4.4.1 Preparative Chromatography ......................................................................... 41 4.4.2 Buffer Exchange and Protein Concentration .............................................. 41 III Contents 4.5 Protein Crystallization ........................................................................................................ 42 4.5.1 Static µL-Scale Crystallization ........................................................................ 42 4.5.2 Stirred mL-Batch Crystallization ................................................................... 43 4.5.3 Crystal Dissolution and Recrystallization.................................................... 45 4.5.4 Calculation of the Crystallization Yield ....................................................... 45 4.5.5 Phase Diagrams .................................................................................................. 46 4.5.6 Crystal Preparation for Neutron and X-Ray Crystallography ................ 46 4.6 Protein and Crystal Analyses ............................................................................................ 47 4.6.1 Protein Concentration Analysis ..................................................................... 47 4.6.2 LbADH Purity Analysis .................................................................................... 47 4.6.3 Enzymatic Activity Assay ................................................................................ 48 4.6.4 Stability Analysis ............................................................................................... 49 4.6.5 Automated Light Microscopy ......................................................................... 50 4.6.6 Structural Analyses ........................................................................................... 50 5 LbADH Process Implementation from Gene to Crystal ...................................... 53 5.1 Choice for LbADH as Exemplary Protein ....................................................................... 53 5.2 Production and Crystallization of LbADH WT ............................................................. 54 5.2.1 Linker Modifications ......................................................................................... 54 5.2.2 Recombinant Production .................................................................................. 55 5.2.3 1-Step Chromatographic Purification ........................................................... 56 5.2.4 Initial Crystallization ........................................................................................ 57 5.3 Crystallization Screenings .................................................................................................. 58 5.3.1 Setup for The Neutron Crystallography Screening ................................... 58 5.3.2 Setup for the Mutant Screening ..................................................................... 60 5.4 Crystallography of LbADH WT ........................................................................................ 61 5.4.1 Neutron Crystallographic Structure .............................................................. 61 5.4.2 X-Ray Crystallographic Structure.................................................................. 61 6 Crystal Contact Engineering of LbADH .................................................................. 63 6.1 Engineering Strategies ........................................................................................................ 63 6.2 Crystal Contacts of LbADH ............................................................................................... 64 6.3 Crystallization of LbADH Variants on the µL-Scale .................................................... 65 6.3.1 Significance and Reproducibility of Varying Crystallization Results ... 65 6.3.2 Characterization of Crystallizability ............................................................. 68 6.3.3 Mutant Screening on the µL-Scale ................................................................. 70 6.3.4 Phase Diagrams of Selected Mutants ............................................................ 74 IV Contents 6.4 Structural Analysis of LbADH Mutants .......................................................................... 76 6.4.1 Crystal Packing .................................................................................................. 76 6.4.2 Altered Crystal Contact Interactions ............................................................ 77 6.5 LbADH Double Mutants – Investigation of Synergetic Effects ................................ 81 6.6 Protein Analysis ................................................................................................................... 82 6.6.1 Enzymatic Activities ......................................................................................... 82 6.6.2 Thermal Stability ............................................................................................... 85 6.6.3 CD Spectra – Secondary Structure Analysis ............................................... 87 6.7 Crystallization Across Different Agents and Buffer Systems ................................... 87 7 Stirred mL-Crystallization of LbADH Variants ..................................................... 89 7.1 Crystallization of Purified Proteins ................................................................................. 89 7.1.1 Reproducibility of Stirred-mL Scale Crystallization .................................. 89 7.1.2 Impact of PEG and Protein Concentration on WT Crystallization