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New gravity-flow column gives faster and simpler purification of histidine-tagged proteins

A. Bergh, M. Carlsson, A. Karlsson, and H. Lindgren
GE Healthcare, Uppsala, Sweden


Histidine-tagged proteins* can be quickly and simply purified on His GraviTrap columns prepacked with Ni Sepharose 6 Fast Flow without the need for a pump or purification system. The protein binding capacity (approx. 40 mg/column) is among the highest available on the market, and separations can be completed in as little as 30 min. Large volumes of clarified or unclarified samples can easily be applied and the purified protein can be eluted in a small volume resulting in a highly concentrated target protein.

Introduction
Immobilized metal ion affinity chromatography (IMAC) is a widely used and highly effective technique that exploits the affinity of histidine-tagged proteins to metal ions attached to chelating separation media, such as Ni Sepharose 6 Fast Flow. His GraviTrap is a single-use column prepacked with Ni Sepharose 6 Fast Flow. His GraviTrap provides high protein binding capacity, short purification times, and simple operation.

The purification of histidine-tagged proteins on His GraviTrap can be divided into four stages: equilibration, sample application, washing, and elution (Fig. 1). Purifications can be performed either under native or denaturing conditions, and a number of additives can be used. In addition, new accessory products further increase speed and convenience.


His GraviTrap and accessories
His GraviTrap contains 1 ml of Ni Sepharose 6 Fast Flow, which delivers a protein binding capacity of approximately 40 mg histidine-tagged protein/column. The columns are made of bio-inert polypropylene, and special frits protect the medium from running dry during use. The column packaging can be converted into a stand (Workmate), and the plastic tray in this package can b e used to collect liquid waste.

The optional LabMate reservoir increases the loading capacity from 10 ml to approximately 35 ml. Large volumes can thus be applied in one go without having to constantly attend the column to add more sample.

The new His Buffer Kit delivers added convenience by providing phosphate buffer concentrates and highly pure 2 M imidazole stock solutions. This kit eliminates time-consuming manual buffer preparation, thereby promoting optimal convenience, performance, and reproducibility.

Faster purifications under a wide range of conditions
The performance of His GraviTrap was compared with Ni-NTA Superflow™ gravity-flow column (Qiagen Inc) during the purification of histidine-tagged green fluorescent protein (GFP-[His]6; Mr 28 000) from an E. coli lysate. The imidazole concentration in the sample and binding buffers was 45 mM for His GraviTrap and 10 mM for Ni-NTA Superflow (the latter according to manufacturer’s recommendations).

The recovery was greater than 98% in the first 3 ml eluate from His GraviTrap, and approximately 80% from the other column. For complete elution from Ni-NTA Superflow, a total volume of 6 to 9 ml was needed, and the total purification time was close to 100 min. The total purification time was 20 min when His GraviTrap was used (Fig 2A). The purity of the eluates was similar as determined by SDS-PAGE (data not shown).

His GraviTrap and Ni-NTA Superflow columns were also compared under denaturing conditions. Histidine-tagged Maltose Binding Protein (MBP-(His)6; Mr 43 000) in E. coli BL-21 lysate containing 7 M urea was applied to the columns. Elution was achieved by lowering the pH from pH 8.0 to pH 4.5. Although denaturing conditions increase the purification time due to the higher viscosity of the samples and buffers, total purification time was still app roximately four times faster on His GraviTrap (Fig 2B).

Complete details of these comparisons are provided in data file 11-0036-90, available at www.amershambiosciences.com/promo_dm205

His GraviTrap also enables rapid separation of large proteins. To demonstrate this, His GraviTrap was used to purify (His)10-TRX-P450 (Mr 133 200). E. coli JM109 cells expressing (His)10-TRX-P450 were enzymatically and mechanically lysed and a 20 ml clarified sample was loaded onto His GraviTrap using LabMate. The whole purification took just 25 min.

Results show that the purification yielded three major protein bands when analyzed by SDS-PAGE (Fig 3A, lane 4). Western blot analysis confirms that each of the three bands contains a histidine-tag (Fig 3B). However, only a weak band can be seen from the band at approximately Mr 15 000. Some of the smaller proteins may have passed through the membrane due to a prolonged blotting time. N-terminal sequencing (data not shown) of the three bands confirms that the low molecular weight bands are truncated forms of the histidine-tagged target protein. To separate the full-length protein from the truncated forms, a second purification step, such as gel filtration, is recommended.


Summary
His GraviTrap columns provide a very fast and simple method for purifying histidine-tagged proteins without the need for a pump or purification system. The prepacked Ni Sepharose 6 Fast Flow gives high protein binding capacity. Proven protocols, application data, and new support products help optimize purification results and simplify the whole procedure.

To obtain data file 11-0036-90, visit www.am ershambiosciences.com/promo_dm205


Acknowledgements
GFP-(His)6 was provided by Dr. David Drew, Dept. of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. MBP-(His)6 was provided by Pharmacia Diagnostics, Uppsala, Sweden.



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