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Purification of MBP-tagged proteins using prepacked columns
A. Bergh1, K. Busson1, M. Carlsson1, A. Heijbel1, A. Karlsson1, J. Lundqvist1, E. M. Maier2, R. Zimmermann3, J. Zou1
1 GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden
2 Dr. von Haunersches Kinderspital, 80337 Munchen, Germany
3University of Leiden, NL 2333CC Leiden, The Netherlands
Sponsored by GE Healthcare
Published in July 2011 (p.17) Sponsored,vendor-submitted protocol
Maltose binding protein (MBP) is beneficial as an affinity tag due to its ability to increase expression level and solubility of the fusion protein. The MBPTrap™ HP 1 ml and 5 ml columns have been specially developed for high specificity towards the MBP-tag and the prepacked format facilitate fast, convenient and reproducible purifications. Additionally, it can easily be regenerated using 0.5 M sodium hydroxide without losing binding capacity.
In this work, MBP-tagged proteins expressed in E. coli were purified on prepacked 1 ml or 5 ml MBPTrap HP columns. A stability study was performed with six repeated purification runs on the same column, each followed by regeneration with 0.5 M NaOH. The results showed no significant change in yield or purity.
A purification of medium-chain acyl-CoA dehydrogenase (MCAD), a protein involved in metabolic disease, was performed. The total purification time was reduced due to elution of highly concentrated target protein, thereby eliminating the need for a concentration step and simplifying the whole purification procedure. Furthermore, an automated two step purification of apoptin was performed on ÄKTAxpress™ where the affinity chromatography step on MBPTrap HP was followed by a gel filtration step.
In summary, the results show that prepacked MBPTrap HP columns decrease purification time and give high purity and reproducibility.
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