Purification of Strep(II)‐tagged proteins using new prepacked columns

Abstract

The Strep(II)‐tag is an ideal tag due to the small size (8 aa, 1 kDa), often making removal of the tag unnecessary. The prepacked StrepTrap™ HP 1 ml & 5 ml columns have been specially developed for high specificity towards the Strep(II)‐tag facilitating fast and convenient purification. Purification is done under physiological conditions and mild elution preserves the activity of the target protein. The 34 um bead size of the resin allows elution in narrow peaks, minimizing the need for further concentration steps.In this work dual‐tagged fluorescent protein, (His)6‐mCherry‐Strep(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. Finally, six repeated purifications of a Strep(II)‐tagged 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, no tendency of decreasing values, demonstrating the high reproducibility.