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Addition of imidazole during binding improves purity of histidine-tagged proteins

K. Hölscher, M. Richter-Roth, and B. Felden de Neumann
GPC Biotech AG, Martinsried, Germany


The purity of histidine-tagged proteins* purified by metal chelate affinity chromatography can often be improved by optimizing the imidazole concentration in the sample and binding buffer to achieve a balance between purity and yield of the protein of interest. We determined a concentration range of imidazole that minimized the nonspecific binding of untagged proteins to Ni Sepharose™ High Performance, thereby greatly improving target protein purity. We demonstrate this approach using histidine-tagged protein kinase G ([His]6-PknG) from Mycobacterium bovis.


Introduction
The (histidine)6 tag is one of the most common tags used to facilitate the purification of recombinant proteins. However, the presence of surface-exposed histidine residues or other complex-forming amino acids can lead to nonspecific binding of untagged host cell proteins to purification media. These untagged proteins elute with the target protein and must be subsequently removed. In general, the binding affinity of these contaminants is lower than that of the tagged recombinant proteins, allowing their separation from the protein of interest by using more stringent conditions.

There are several ways to reduce the binding of contaminating proteins to Ni Sepharose media. On one hand, the amount of Ni Sepharose can be adapted to the expected amount of protein to be purified. On the other hand, imidazole can be used as a competitive agent. In this article, we demonstrate the importance of imidazole as a useful tool to enhance the purity of (His)6-PknG purified with Ni Sepharose High Performance.


Determination of the optimal imidazole concentration
To determine the optimal imidazole concentration to use du
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