Thesis final - after defense-7 - Jacobs University

Table of Contents
1. Introduction ............................................................................................................................ 1
1.1. Introduction to Biotechnology ........................................................................................ 1
1.2. Downstream processing .................................................................................................. 2
1.3. Hydrophobic interaction chromatography (HIC) ............................................................ 4
1.4. Factors affecting protein chromatography behavior in HIC ........................................... 7
1.4.1. Effect of mobile phase parameters ........................................................................... 7
1.4.2. Effect of stationary phases in HIC ......................................................................... 10
1.4.3. Effect of protein properties in HIC ........................................................................ 12
1.4.4. The comparison between HIC and RPC ................................................................ 13
1.5. Analysis of the yeast proteome as biotech feedstock .................................................... 15
1.6. Mass spectrometry and database searching ................................................................... 16
1.7. The in silico Approach .................................................................................................. 18
1.8. Goal of the work ............................................................................................................ 21
2. Experimental approach ......................................................................................................... 24
2.1. Materials ........................................................................................................................ 24
2.2. Experimental strategy .................................................................................................... 24
2.2.1. Cell Cultivation ...................................................................................................... 26
2.2.2. Preparation of the crude extract ............................................................................. 26
2.2.3. Chromatographic fractionation .............................................................................. 26
2.2.4. Membrane dialysis ................................................................................................. 27
2.2.5. Two dimensional polyacrylamide gel electrophoresis (2-D PAGE) ...................... 27
2.2.6. Protein content of the fractions .............................................................................. 29
2.2.7. Protein identification by MALDI-ToF-MS ............................................................ 29
2.2.8. Characterization of proteins utilizing Bioinformics ............................................... 31
3. Results and Discussion ......................................................................................................... 35
3.1. Influence of the different ligand chemistries during hydrophobic interaction
chromatography ........................................................................................................................ 35
3.1.1. Summary .................................................................................................................... 35
3.1.2. Scientific background of the approach ....................................................................... 36
3.1.2.1. Hydrophobic adsorbents ...................................................................................... 36
3.1.2.2. Protein properties in the context of HIC ............................................................. 39
3.1.2.3. Towards an in silico approach for the downstream processing of bioproducts .. 43
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