Purification of Histidine-Tagged Proteins using HiTrap® TALON® Crude

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

HiTrap® TALON® crude is 1 ml and 5 ml HiTrap® columns prepacked with TALON® Superflow. The columns can be operated with either a syringe, a peristaltic pump, or a chromatography system such as ÄKTA. After thorough cell disruption, it is possible to load the unclarified lysate on the specially designed column without precentrifugation and filtration.

The HiTrap® TALON® crude column is made of biocompatible polypropylene that does not interact with biomolecules. Columns are delivered with a stopper on the inlet and a snap-off end on the outlet. See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON® Superflow, and uncharged IMAC Sepharose products) for the main characteristics of HiTrap® TALON® crude.


Sample and buffer preparation

Refer to Purification using TALON® Superflow earlier in this chapter for a general procedure for sample and buffer preparation.


Purification

  1. Fill the pump tubing or syringe with distilled water. Remove the stopper and connect the column to the chromatography system tubing, syringe (use the adapter provided), or laboratory pump “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 or 5 ml/min for the 1 ml and 5 ml columns, respectively. In some cases, a blank run is recommended before final equilibration/sample application.

Leakage of Co2+ from TALON® Superflow is low under all normal conditions. For very critical applications, leakage during purification can be even further reduced by performing a blank run before loading sample. See next page for blank run.

  1. Apply the unclarified lysate with a pump or a syringe. Continuous stirring of the sample during sample loading may be necessary to prevent sedimentation.
    Typical loading volumes of unclarified lysate (highly dependent on specific sample, sample pretreatment, and temperature at sample loading):
    HiTrap® TALON® crude 1 ml: Up to 100 ml
    HiTrap® TALON® crude 5 ml: Up to 500 ml

Sample loading at 4°C may increase the viscosity of the sample. An adverse effect of increased sample viscosity is that maximum back pressure for the column is reached at a lower sample volume loading on the column. Do not exceed the maximum back pressure of the column. Large volumes may increase back pressure, making the use of a syringe more difficult.

  1. Wash with wash buffer until the absorbance reaches a steady baseline (generally at least 15 to 20 column volumes).
  2. Elute with elution buffer using a one-step procedure or a linear gradient.
    1. For step elution, 8 column volumes of elution buffer are usually sufficient. A shallow gradient, for example, a linear gradient over 20 column volumes or more, can separate proteins with similar binding strengths.

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.


Blank run:

  1. Wash the column with 5 column volumes of distilled water.
  2. Wash with 5 column volumes of elution buffer.
  3. Equilibrate with 10 column volumes of binding buffer.

 

Application example

Yield and purity using unclarified vs clarified sample with HiTrap® TALON® crude

HiTrap® TALON® crude columns simplify purification by eliminating the need for precentrifugation and filtration steps. For comparison, unclarified and clarified (centrifugation at 30 000 × g for 20 min) E. coli extracts containing the histidine-tagged protein GEHC1-(His)6 were loaded on HiTrap® TALON® crude 1 ml columns. After purification, purity (analyzed by SDS-PAGE) and yield (calculated from absorbance measurements) of eluted fraction pools were determined.

Figure 3.44 shows that purification using unclarified extract was similar to when clarified sample was used. The amount of GEHC1-(His)6 eluted was 13 mg and 12 mg when loading unclarified and clarified sample, respectively. In addition, the purity did not significantly differ, and SDS-PAGE  analysis showed high purity for both samples (> 90%; Fig 3.45). By eliminating the need for precentrifugation and filtration, HiTrap® TALON® crude columns saved approximately 40 min.

Comparison study

Fig 3.44. Comparison study loading unclarified (blue line) and clarified (orange line) E. coli samples containing the histidine-tagged protein GEHC1-(His)6 on HiTrap® TALON® crude. Overlay of absorbance curves.

 

SDS-PAGE analysis

Fig 3.45. SDS-PAGE analysis (reducing conditions, ExcelGel SDS Gradient 8-18, Coomassie stained) of pools containing purified GEHC1-(His)6. Comparison of runs performed on HiTrap® TALON® crude using unclarified and clarified sample.

Materials

     
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