Tagged protein purification

What is tagged protein purification?

Tagged protein purification uses affinity chromatography to purify recombinant proteins that have been engineered to include a specific peptide or protein sequence (tag). Adding a tag to a target protein allows you to simplify the purification protocol greatly, sometimes to the extent that you can use a standard protocol.

Common choices for affinity tags are polyhistidine (histidine-tag), Glutathione S-transferase (GST), maltose-binding protein (MBP), Strep-tag II and FLAG™.

How does tagged protein purification work?

The target protein with affinity tag is specifically and reversibly bound by a complementary binding substance (ligand) that recognizes the tag. The sample is applied under conditions that favor binding to the ligand. Unbound material is washed out of the column.

The bound tagged protein is recovered (eluted), commonly using a competitive ligand. The eluted protein is usually at a high concentration. If tag removal is needed prior to use of the protein, cleavage can be performed using a site-specific protease.

Tagged_protein_purification_landscape

When should I use tagged protein purification?

Affinity purification of tagged proteins can be used as the only purification step for applications that do not require proteins with extremely high purity.

When the highest purity is needed, this technique can be used as the first (capture) step in a multistep purification procedure. Another option is to produce proteins with a different tag on the 5’ and 3’ ends (dual-tagged proteins). Purify using affinity resins for one of the tags, then purify using resins for the other tag.

Which affinity tag should I choose?

Adding an affinity tag to your protein can simplify purification and detection and can improve solubility and stability. But if a tag could interfere with use of your protein, you might consider either removing it after purification or purifying the native protein instead.

If you do decide to add a tag, considerations for choosing the appropriate tag should include the purification priorities, size of the tag, and cost of the chromatography resins.


  His Strep-tag™ II GST MBP FLAG™
Size 6 aa 8 aa 26 kDa 40 kDa 8 aa
Protein binding capacity 40 mg/mL 6 mg/mL 30 mg/mL 10 mg/mL 0.6 mg/mL
Purity + +++ ++ ++ +++
Increased solubility No No Yes Yes No
Risk for interference with function Low Low High High Low

How can I remove the affinity tag from my protein?

Your tagged protein must include a recognition sequence for the protease that you plan to use.

Cleavage can be done on the column or after the tagged protein has been eluted. Common proteases with low specificity are thrombin and factor Xa. Proteases with higher specificities are available. Recombinant proteases that have the same tag as the target protein can be removed with the same resin that was used to purify the target.

scissors_explanation

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