Process development for the control of solubility of Affibody® molecules
نویسنده
چکیده
................................................................................................................................................ 1 1 BACKGROUND ...................................................................................................................................... 3 1.1 INTRODUCTION ................................................................................................................................................... 3 1.1.1 Affibody® molecules ................................................................................................................................ 3 1.1.2 Expression systems ................................................................................................................................... 4 1.1.3 Fermentation methods ........................................................................................................................... 5 1.1.4 MODDE .......................................................................................................................................................... 6 1.1.5 Target proteins .......................................................................................................................................... 6 2 MATERIALS AND METHODS ............................................................................................................ 7 2.1 STRAINS AND PLASMIDS .................................................................................................................................... 7 2.2 TRANSFORMATION ............................................................................................................................................. 8 2.3 WORKING CELL BANK (WCB) ......................................................................................................................... 8 2.4 MEDIUM ............................................................................................................................................................... 8 2.5 CULTIVATION ...................................................................................................................................................... 8 2.6 PROTEIN EXPRESSION ........................................................................................................................................ 9 2.7 EXPRESSION ANALYSIS ...................................................................................................................................... 9 2.7.1 Quantification ........................................................................................................................................... 10 2.8 PURIFICATION .................................................................................................................................................. 10 2.9 PROTEIN ANALYSIS .......................................................................................................................................... 10 2.10 OPTIMIZATION .............................................................................................................................................. 11 3 RESULTS .............................................................................................................................................. 12 3.1 EXPRESSION OF PAY02631 IN LEMO2 (DE3) AND BL21(DE3) ........................................................ 12 3.1.1 Characterization of Lemo21(DE3) and determination of the optimal rhamnose concentration ....................................................................................................................................................... 12 3.2.1 Fed-‐batch cultivation in BL21(DE3) ............................................................................................... 13 3.3 EXPRESSION OF PAY02610 IN LEMO21(DE3) AND BL21(DE3) ...................................................... 13 3.3.1 Characterization of Lemo21(DE3) and determination of the optimal rhamnose concentration ....................................................................................................................................................... 13 3.3.2 SDR (PreSens) ........................................................................................................................................... 14 3.3.3 Optimization in Lemo21(DE3) .......................................................................................................... 15 3.4 Optimization in BL21(DE3) ................................................................................................................... 16 3.4.1 Protein purification ................................................................................................................................ 16 3.4.1 Optimization (MODDE) ........................................................................................................................ 17 4 DISCUSSION ....................................................................................................................................... 19 5 REFERENCES ...................................................................................................................................... 21
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