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Purification of Strep-tag™ II proteins using prepacked columns
A. Bergh1, M. Carlsson1, A. Heijbel1, U. Hjellström-Nilsson1, A. Karlsson1, J. Öhman, M. Nilsson2
1 GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden
2 Biovitrum, SE-112 87 Stockholm, Sweden
Sponsored by GE Healthcare
Published in July 2011 (p.19) Sponsored,vendor-submitted protocol
The Strep-tag II is ideal as an affinity tag due to the small size (8 aa, 1 kDa), often making removal of the tag unnecessary.
StrepTrap™ HP 1 ml and 5 ml columns have been specially developed for high specificity towards the Strep-tag II, with the prepacked format facilitating fast and convenient purification. Purification is done under physiological conditions and the mild elution preserves the activity of the target protein. The small bead size of the medium (34 m) allows elution in narrow peaks, minimizing the need for further concentration steps.
In this work, purifications of Strep-tag II proteins on StrepTrap HP 1 ml and 5 ml columns are presented. A dual-tagged fluorescent protein, (His)6-mCherry-Strep-tag II expressed in E. coli, was purified in a purification procedure including two affinity columns, StrepTrap HP followed by HisTrap™ HP. The second affinity column was necessary for removal of a truncated variant of the target protein. The multi-step purification was performed fully automatically using ÄKTAxpress™. Another dual-tagged protein expressed in insect cells was purified to high purity in a single step using StrepTrap HP. The final purity was above 95% according to SDS-PAGE analysis.
Finally, six repeated purifications of a Strep-tag II protein expressed in E. coli were performed on the same StrepTrap HP column with 0.5 M NaOH regeneration between the runs. The final purity and yield of the target protein remained almost unchanged, with no tendency of decreasing values, demonstrating the high reproducibility during repeated use of StrepTrap HP.
In summary, the use of prepacked StrepTrap HP columns for convenient purification of Strep-tag II proteins results in high final purity and yield of the target protein. In addition the column is efficiently regenerated with 0.5 M NaOH.
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