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Polymers 2018, 10(5), 468; https://doi.org/10.3390/polym10050468

Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA
These authors contribute equally to this work.
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Received: 6 March 2018 / Revised: 18 April 2018 / Accepted: 20 April 2018 / Published: 25 April 2018
(This article belongs to the Special Issue Polymers for Bioseparations)
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Abstract

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. View Full-Text
Keywords: non-chromatographic protein purification; aggregating tags; split intein; elastin-like polypeptide (ELP); β-roll tag (BRT17); self-cleaving tag non-chromatographic protein purification; aggregating tags; split intein; elastin-like polypeptide (ELP); β-roll tag (BRT17); self-cleaving tag
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Fan, Y.; Miozzi, J.M.; Stimple, S.D.; Han, T.-C.; Wood, D.W. Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags. Polymers 2018, 10, 468.

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