Purification Using HisTrap™ HP and HisTrap™ FF

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

HisTrap HP and HisTrap FF are 1 ml and 5 ml HiTrap columns packed with Ni Sepharose High Performance or Ni Sepharose 6 Fast Flow, respectively. Sample application, washing,  and elution can be performed using a syringe with a supplied adapter, a peristaltic pump, or a liquid chromatography system such as ÄKTA (see Chapter 2, Manual and automated purification, Table 2.1 for equipment choices).

HisTrap HP and HisTrap FF columns are made of polypropylene, which is biocompatible and non-interactive with biomolecules. The top and bottom frits are manufactured from porous polyethylene. Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. Every package includes all necessary components for connection of the columns to different types of equipment. For quick scale-up of purifications, two or three HisTrap columns (1 ml or 5 ml) can be connected in series (back pressure will be higher). Note that HisTrap HP and HisTrap FF columns cannot be opened or refilled.

HisTrap HP and HisTrap FF 1 ml and 5 ml columns

Fig 3.11. HisTrap HP and HisTrap FF 1 ml and 5 ml columns allow convenient and simple one-step purification of histidine-tagged proteins. HisTrap HP 1 ml and 5 ml columns are shown here. The simple purification scheme is shown at right.


Sample and buffer preparation

HisTrap HP: refer to Purification using Ni Sepharose High Performance earlier in this chapter for a general procedure for sample and buffer preparation.

HisTrap FF: refer to Purification using Ni Sepharose 6 Fast Flow earlier in this chapter for a general procedure for sample and buffer preparation.


Purification

  1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied), laboratory pump, or chromatography system “drop to drop” to avoid introducing air into the system.
  2. Remove the snap-off end at the column outlet.
  3. Wash out the ethanol with 3 to 5 column volumes of distilled water.
  4. Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min (1 ml column) and 5 ml/min (5 ml column).
  5. Apply the pretreated sample using a syringe fitted to the Luer connector or by pumping it onto the column. For best results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml column) during sample application1.
  6. Wash with binding buffer (generally at least 5 to 10 column volumes) until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for washing.
  7. Elute with elution buffer using a one-step or linear gradient. For step elution, 5 column volumes is usually sufficient. For linear gradient elution, 10 to 20 column volumes is usually sufficient. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for elution.
  8. After elution, regenerate the column by washing it with at least 5 column volumes of binding buffer. The column is now ready for a new purification.

1 One ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap 1 ml column, and 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap 5 ml column.

The column does not need to be stripped and recharged between each purification if the same protein is going to be purified. Reuse of any purification column depends on the nature of the sample and should only be performed with identical tagged proteins to prevent cross-contamination. For more information on this topic and on cleaning and storage, refer to Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products).

Ni Sepharose is compatible with reducing agents. However, we recommend removal of any weakly bound Ni2+ ions before applying buffer/sample that includes reducing agents. This can be accomplished by performing a blank run without reducing agents (see below). Do not leave or store HisTrap columns with buffers that include reducing agents.

Leakage of Ni2+ from Ni Sepharose is low under all normal conditions. For very critical applications, leakage during purification can be even further diminished by performing a blank run (as described below) before loading sample.


Blank run:

  1. Use binding buffer and elution buffer without reducing agents.
  2. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
  3. Wash with 5 column volumes of elution buffer.
  4. Equilibrate with 10 column volumes of binding buffer.

Application examples

1. Two-step purification of a high-molecular-weight histidine-tagged protein using HisTrap HP

The high-molecular-weight protein histidine-tagged mannanase Man 26A from Cellulomonas fimi (Mr 100 000) was purified in its enzymatically active form using a 1 ml HisTrap HP column (Fig 3.12A). A second purification step using SEC with Superdex™ 200 (24 ml column volume) was added to obtain a purity of 95% (Figs 3.12B and 3.12C).

First purification step, AC, using HisTrap HP 1 ml column.

(B) Second purification step with SEC using Superdex 200 30/100 GL. (C) SDS-PAGE.

Fig 3.12. (A) First purification step, AC, using HisTrap HP 1 ml column. (B) Second purification step with SEC using Superdex 200 30/100 GL. (C) SDS-PAGE.

 

2. Scaling up purification from HisTrap FF to larger scale

Histidine-tagged maltose binding protein MBP-(His)6 was purified from an E. coli extract. Samples containing 8, 40, and 160 mg, were loaded on a 1 ml HisTrap FF column, a 5 ml HisTrap FF column, and a 20 ml HisPrep FF 16/10 column respectively, all of which were run at the same flow velocity. The results show that scaling up the column dimension while running at the same flow velocity provides highly consistent results (Fig 3.13A–C). Pooled fractions were analyzed by SDS-PAGE and showed almost identical results in terms of purity and recovery (Fig 3.13D).

Scale-up from (A) HisTrap FF 1 ml

 

scale-up via (B) HisTrap FF 5 ml to (C) HisPrep 16/10 (20 ml) prepacked column

Figure 3.13. Scale-up from (A) HisTrap FF 1 ml via (B) HisTrap FF 5 ml to (C) HisPrep 16/10 (20 ml) prepacked column. The samples loaded contained approximately 8, 40, and 160 mg of MBP-(His)6, respectively. Recovery in milligrams is shown in each chromatogram. (D) SDS-PAGE (ExcelGel SDS Gradient 8–18) under nonreducing conditions confirms that scaling up from the 1 ml to the 20 ml column does not significantly affect the purification result.

 

Materials

     
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