Navigation Links
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.

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 during the binding of (His)6-PknG to Ni Sepharose High Performance, a cell lysate containing (His)6-PknG was loaded on a column with 10 mM imidazole in the binding buffer. After additional washing with binding buffer, (His)6-PknG was eluted using a linear gradient of up to 50% elution buffer (250 mM imidazole) within 20 column volumes (CV). By analyzing the collected fractions, it was found that most of the contaminants eluted in the range of about 40 to 70 mM imidazole (data not shown).

Improved purity
Based on this finding, 45 mM imidazole was used in the sample and binding buffer during the final purification procedure. To achieve a higher protein concentration, the protein was eluted in a two-step gradient (Fig 1). To demonstrate the advantageous effect of imidazole, an additional purification was performed under the same conditions except that imidazole was omitted from the sample and binding buffer (Fig 2). SDS-PAGE of the pooled elution fractions indicated a large improvement in purity when 45 mM imidazole was included in the sample and binding buffer (Fig 3).

Using a linear gradient, we determined a concentration range of imidazole that resulted in the removal of a majority of contaminating proteins, while retaining (His)6PknG. The addition of 45 mM imidazole to the sample and binding buffer resulted in much higher purity of the desired protein.

To obtain the Ni Sepharose High Performance data file (18-1174-40) visit

back to top


Page: All 1 2

Related biology technology :

1. PCR Grade Deoxynucleotides Additional power to perfect your PCR experiments
2. The measurement of DNA condensation and mitochondrial changes during apoptosis and necrosis in cell based assays
3. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
4. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
5. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
6. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
7. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
8. Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
9. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System
10. The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System
11. Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:

(Date:10/11/2017)... (PRWEB) , ... October 11, 2017 , ... Disappearing forests ... the lives of over 5.5 million people each year. Especially those living in larger ... startup Treepex - based in one of the most pollution-affected countries globally - decided ...
(Date:10/10/2017)... ... October 10, 2017 , ... For ... has won a US2020 STEM Mentoring Award. Representatives of the FirstHand program travelled ... Volunteer Experience from US2020. , US2020’s mission is to change the trajectory of ...
(Date:10/10/2017)... ... October 10, 2017 , ... The ... prestigious awards honoring scientists who have made outstanding contributions to analytical ... during Pittcon 2018, the world’s leading conference and exposition for laboratory science, which ...
(Date:10/9/2017)... , ... October 09, 2017 ... ... on October 5, 2017, in the medical journal, Epilepsia, Brain Sentinel’s SPEAC® ... gold standard, video EEG, in detecting generalized tonic-clonic seizures (GTCS) using surface ...
Breaking Biology Technology:
(Date:7/20/2017)... DAL ) customers now can use fingerprints instead of their ... (DCA). ... launches biometrics to board aircraft at Reagan Washington National Airport ... Delta,s biometric boarding pass experience that launched in May at ... process to allow eligible Delta SkyMiles Members who are enrolled in CLEAR ...
(Date:6/14/2017)... 15, 2017  IBM (NYSE: IBM ) is introducing several ... dedicated to developing collaboration between startups and global businesses, taking ... During the event, nine startups will showcase the solutions they ... industries. France is ... with a 30 percent increase in the number of startups ...
(Date:5/16/2017)... , May 16, 2017   Bridge ... health organizations, and MD EMR Systems , ... development partner for GE, have established a partnership ... Portal product and the GE Centricity™ products, including ... EMR. These new integrations will ...
Breaking Biology News(10 mins):