| Autor: |
Fan Y; William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. fan.457@osu.edu., Miozzi JM; William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. miozzi.5@osu.edu., Stimple SD; William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. stimple.1@osu.edu., Han TC; William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. tzuchianghan@gmail.com., Wood DW; William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA. wood.750@osu.edu. |
| Abstrakt: |
Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant drawback, however, is that current proteolytic approaches for post-purification tag removal after are expensive and non-scalable. To address this problem, we have developed two non-chromatographic purification strategies that use either the elastin-like polypeptide (ELP) tag or the β-roll tag (BRT17) in combination with an engineered split intein for tag removal. The use of the split intein eliminates premature cleavage during expression and provides controlled cleavage under mild conditions after purification. These self-cleaving aggregating tags were used to efficiently purify β-lactamase (β-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), resulting in increased yields compared to previous ELP and BRT17-based methods. Observed yields of purified targets for both systems typically ranged from approximately 200 to 300 micrograms per milliliter of cell culture, while overall recoveries ranged from 10 to 85 percent and were highly dependent on the target protein. |
