Development of a refolding and capture protocol for HRP IBs produced in E. coli

Abstract

........................................................................................................................................................ 3 Danksagung ................................................................................................................................................... 4 List of Abbreviations ...................................................................................................................................... 6 Introduction ................................................................................................................................................... 7 Methods and Materials ................................................................................................................................. 9 1. Small scale DoEs .............................................................................................................................. 10 1.a Solubilization ................................................................................................................................. 10 1.b Refolding........................................................................................................................................ 11 1.c Redox system ................................................................................................................................. 12 2. Refolding Reactor ............................................................................................................................ 13 2.a Batch refolding .............................................................................................................................. 14 2.b Fed batch refolding ....................................................................................................................... 15 2.c Hemin addition .............................................................................................................................. 15 3. Capture and Concentration ............................................................................................................. 16 3.a Salt precipitation ........................................................................................................................... 16 3.b Chromatography ........................................................................................................................... 16 Analytics .................................................................................................................................................. 17 Results and Discussion ................................................................................................................................ 18 1. Small scale DoEs .............................................................................................................................. 19 1.a Solubilization ................................................................................................................................. 20 1.b Refolding........................................................................................................................................ 22 1.c Redox system ................................................................................................................................. 26 2. Refolding Reactor ............................................................................................................................ 34 2.a Batch refolding .............................................................................................................................. 35 2.b Fed batch refolding ....................................................................................................................... 37 2.c Hemin addition .............................................................................................................................. 40 3. Capture and Concentration ............................................................................................................. 42 3.a Salt precipitation ........................................................................................................................... 42 3.b Chromatography ........................................................................................................................... 44 Conclusion of this Thesis ............................................................................................................................. 48 References ................................................................................................................................................... 50