Condition Screening for Scaling up using HiScreen™ Ni FF

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

HiScreen Ni FF is a ready-to-use column for purification of histidine-tagged recombinant proteins by IMAC. The column has a volume of 4.7 ml of Ni Sepharose 6 Fast Flow and is well suited for screening of selectivity and binding/elution conditions for scaling up, as well as for small-scale purifications. Ni Sepharose 6 Fast Flow has low Ni2+ leakage and is compatible with a wide range of additives used in protein purification. The columns are made of biocompatible polypropylene that does not interact with biomolecules. See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products) for the main characteristics of HiScreen Ni FF.

The columns are used in an optimal way with liquid chromatography systems such as ÄKTA.

HiScreen Ni FF column

Fig 3.24. HiScreen Ni FF column for establishing optimal chromatographic conditions for scaling up.

 

Sample and buffer preparation

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

Purification

The recommended flow rate for HiScreen Ni FF is 2.3 ml/min (300 cm/h flow velocity).

  1. Equilibrate with at least 5 column volumes of binding buffer. Avoid introducing air into the column.
  2. Adjust the sample to the chosen starting conditions and load on the column.
  3. Wash with 5 to 10 column volumes of binding buffer until the UV trace of the effluent returns to near baseline.
  4. Elute either by linear gradient elution or step elution at recommended flow rates. If required, the collected eluted fractions can be buffer-exchanged or desalted.
    Linear gradient elution: Elute with 0% to 100% elution buffer (up to 500 mM imidazole) in 10 to 20 column volumes.
    Step elution: Elute with 5 column volumes of elution buffer including imidazole at chosen concentration. Repeat at higher imidazole concentrations until the target protein has been eluted.
  5. If required, strip the column from metal ions and perform cleaning-in-place (CIP) to clean the column.
  6. Re-equilibrate the column with 5 to 10 column volumes of binding buffer or until the UV baseline, eluent pH, and conductivity reach the required values.

Do not exceed the maximum recommended flow and/or back pressure for the column.

For A280 measurements, use the elution buffers as blanks. If imidazole needs to be removed, use a desalting column (see Chapter 11, Desalting/buffer exchange and concentration). Low-quality imidazole will give a significant background absorbance at 280 nm.

In some cases, we recommend a blank run before final equilibration/sample application, as described below. Perform a blank run without reducing agents before applying buffers/samples containing reducing agents. Likewise, a blank run is recommended for critical purifications where metal ion leakage during purification must be minimized.

Blank run:

Use binding buffer and elution buffer without reducing agents.

  1. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
  2. Wash with 5 column volumes of elution buffer.
  3. Equilibrate with 10 column volumes of binding buffer.

 

Application example

Purification of GFP-(His)6 expressed in E. coli

HiScreen Ni FF was used for purification of histidine-tagged green fluorescent protein, GFP-(His)6, expressed in E. coli. The elution was performed using a 10 column volume linear gradient with imidazole up to 500 mM. Two partly resolved peaks were obtained; absorbance measurement at the specific wavelength 490 nm indicated the presence of GFP-(His)6 in the second peak (Fig 3.25).

This finding was confirmed by SDS-PAGE analysis: the second pool contained highly pure GFP-(His)6, > 95% whereas the first peak, Pool 1, contained primarily contaminants (Fig 3.26). The amount of GFP-(His)6 in the second pool was 42 mg as determined by measurement of absorbance at 490 nm.

 Purification of GFP-(His)6

Fig 3.25. Purification of GFP-(His)6 expressed in E. coli BL21 on HiScreen Ni FF. Indicated pools were analyzed by SDS-PAGE (Fig 3.26).

 

SDS-PAGE analysis

Fig 3.26. SDS-PAGE analysis (reducing conditions) of GFP-(His)6 after purification on HiScreen Ni FF.  ExcelGel SDS Gradient 8-18.

Materials

     
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