Tagged for success – but not forever Affinity tag removal of tagged-proteins

Introduction

The majority of proteins purified in laboratory scale are affinity tagged, with e.g., His, GST, or MBP tag, and can therefore be purified with relative ease using one affinity chromatography step. Affinity tags greatly enhance the efficiency and flexibility of protein purification, making it easier to obtain high-quality, functional proteins for a wide range of research and industrial applications.

In some case the presence of a tag on a purified protein can affect its function, structure, stability, or interaction with other molecules, the tags need therefore to be removed in these cases. Usually, a protease cleavage site is included in the protein sequence, so the tag can be removed using a specific protease, such as thrombin or Factor Xa, from the purified protein.

The removal of tag can be carried out using either batch or on-column cleavage. After cleavage, cleaved tag, uncleaved proteins, and protease need to be removed to obtain pure target protein without the tag.

The amount of protease, temperature, and length of incubation required for complete digestion varies according to the nature of the target protein. The amount of protease needed is usually measured in international units, not in micrograms, of a protease. One unit is defined as the amount of the protease that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method, i.e., a measure of the protease’s catalytic activity. Follow the instructions for use to add suitable amount of protease for cleavage of your tagged protein. Since thrombin is a disulfide-linked heterodimer it is sensitive to reducing agents, while TEV (tobacco etch virus) protease and PreScission Protease can be used in the presence of reducing agents such as DTT (Dithiothreitol).

There are two ways to carry out tag removal: tag removal with on-column cleavage or tag removal with cleavage in solution. In general, a larger amount of enzyme is needed for on-column cleavage using e.g. PreScission™ protease—which is GST-tagged with a prepacked GSTrap™ column—than for tag cleavage in solution, since the resin is more fixed in a prepacked column and the access to the GST-tagged protein is more limited for the enzyme. To be able to reach set incubation time and recovery, additional enzymes are often needed for the on-column cleavage.

An alternative to purification using traditional affinity tag and tag cleavage with protease is to use the Cytiva™ Protein Select™ resin. This resin is an affinity chromatography resin for purifying recombinant proteins using the self-cleaving Cytiva Protein Select tag. During an affinity step performed with Cytiva Protein Select resin, the protein self-cleaves from the tag and elutes with no residual tag amino acids.

1. Tag removal with on-column cleavage

A convenient and automated option is to remove both the tag and the protease using on-column cleavage and a chromatography system.

Different approaches are possible depending on the protease used. Non-tagged proteases such as thrombin or Factor Xa can be removed using a benzamidine resin. In benzamidine Sepharose™ FF, p-Aminobenzamidine (pABA) a synthetic inhibitor of trypsin and trypsin-like serine proteases, is covalently coupled to Sepharose 4 Fast Flow via a stable linkage. The resin has high capacity for trypsin and trypsin-like serine proteases such as thrombin. The binding capacity for trypsin is 35 mg trypsin/mL resin. Trypsin-like serine proteases bind to benzamidine Sepharose resin at neutral pH (e.g., with a buffer of 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4). The bound protease molecules can be eluted from the resin using a buffer of 50 mM glycine, pH 3.0.

Tagged proteases can be removed with the same affinity chromatography resin and/or column used to capture the tagged protein of interest. For example, histidine-tagged TEV proteases are commonly used to cleave tags from histidine-tagged target proteins and can be removed by an immobilized metal affinity chromatography (IMAC) column. In addition, GST-tagged human rhinovirus 3C (HRV3C) proteases, such as PreScission protease, can be used to cleave GST-tagged proteins and removed by a glutathione affinity column.

To minimize handling and maximize speed in all the procedures, we suggest attaching columns to a chromatography system such as an ÄKTA™ system. Purification can also be performed using a syringe or a simple pump.

1.1 Remove tag and non-tagged proteases: thrombin or Factor Xa

GST-tagged proteins: remove tag and thrombin or Factor Xa

1. Isolate your GST-tagged protein using a glutathione affinity column, such as GSTrap 4B column (prepacked with Glutathione Sepharose 4B resin). Wash out unbound protein with binding buffer. Equilibrate the column with the optimal cleavage buffer for thrombin or Factor Xa.
2. Inject the protease into the GSTrap column and seal both ends.
3. Incubate for 2 hours (or a suitable time for an optimal yield of cleaved target protein) at room temperature.
   During the incubation, equilibrate a HiTrap™ Benzamidine FF (High Sub) column (prepacked with Benzamidine Sepharose Fast Flow) with the recommended binding buffer. Connect the GSTrap column on top of the HiTrap Benzamidine column.
4. After incubation is complete, wash the connected columns with binding buffer.

This procedure allows the cleaved protein and thrombin or Factor Xa to be washed out from the GSTrap 4B column. As the sample passes through the connected HiTrap Benzamidine column, the protease binds; however, the de-tagged protein passes through the column for collection in the flowthrough.

1.2 Remove tag and tagged proteases

GST-tagged proteins: remove tag and GST-tagged protease

PreScission protease is a fusion protein consisting of GST and human rhinovirus (HRV) type 14-3C protease. Since the protease is GST-tagged, it is easily removed from cleavage reactions using Glutathione Sepharose resin.

It is suggested to use the following buffer for cleavage using PreScission protease:

50 mM Tris-HCl, 150 mM NaCl; 1 mM EDTA; 1 mM DTT, pH 7.0. The cleavage should be carried out at temperature between 5-15°C.

The cleavage of PreScission Protease may be improved by adding detergents (such as Tween 20or NP40) to a concentration of 0.01%. Concentrations of these detergents up to 1% do not inhibit PreScission Protease. The presence of Zn2+ as well as other inhibitors (e.g., Chymostati or Pefabloc SC) will interfere with the activity of PreScission Protease.

The following procedure uses the same affinity column for target protein purification and protease removal.

1. Isolate your GST-tagged protein using a glutathione affinity column, such as GSTrap 4B column (prepacked with Glutathione Sepharose 4B resin). Wash out unbound protein with binding buffer. Then equilibrate the column with the optimal cleavage buffer for PreScission protease.
2. Inject GST-tagged PreScission protease and seal the column.
3. Incubate for 2 hours at room temperature.
4. After incubation, restart the flow and wash the column with cleavage buffer.

The de-tagged protein will pass through and is collected. The GST-tagged protease, cleaved GST-tag, and any uncleaved target protein will remain bound on the column.

The following is an example of purification and on-column cleavage of GST-tagged TLP40 protein using GSTrap FF columns and PreScission Protease

For PreScission Protease digestion, 2 units of enzyme/100 µg of bound GST-tagged protein. The cleavage buffer contains 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0. The on-column cleavage time is 12 to 16 hours at 4°C.

Prior to elution, a 1 mL GSTrap FF column was connected downstream to the GSTrap FF proteolytic cleavage columns to capture any released GST, uncleaved GST-tagged protein, and unbound PreScission Protease, whereas the cleaved protein was directly eluted.

Figure 1B shows a pure TLP40 protein without the GST tag (lane 7) was obtained after this step.

Fig 1. Purification and SDS-PAGE analysis of TLP40-GST-tagged protein. (A) Purification and on-column cleavage of tagged protein using GSTrap FF 5 mL and PreScission Protease (B) Fractions from the purification steps were analyzed by SDS-PAGE.

2. Tag removal with cleavage in solution

2.1 Remove His tag and His-tagged proteases

His-tagged proteins: remove tag and his-tagged protease

The following procedure uses the same affinity column for target protein purification and protease removal.

1. Isolate your his-tagged protein using a nickel- or cobalt-charged IMAC column, such as HisTrap HP or HiTrap TALON crude, respectively. Wash out unbound protein with binding buffer and elute your protein with imidazole-containing buffer. Then equilibrate the column with IMAC binding buffer.
2. Exchange the buffer from IMAC elution buffer to the optimal buffer for cleavage with his-tagged TEV protease using a dialysis tube or desalting column (e.g., PD-10 desalting columns packed with Sephadex G-25 resin).
3. Incubate for 15 hours at 4°C, which is the optimal cleavage temperature for TEV protease.
4. After incubation, loading the solution to the same IMAC column.

The de-tagged protein will pass through and is collected. The his-tagged protease, cleaved his-tag, and any uncleaved target protein will remain bound on the column.

The following is an example of purification of His-tagged Interleukin-1β (IL-1β) containing TEV cleavage site and subsequent tag cleavage and removal. The solutions are analyzed with SDS-PAGE. 1 mg of TEV protease was added to a solution containing 24 mg of His-tagged protein. The cleavage occurs at 4°C overnight in a buffer containing 30 mM Tris, 150 mM NaCl at pH 7.5.

The SDS-PAGE shows a pure final product (lane 6) at molecular weight corresponding to the protein without the His tag.

Fig 2. SDS-PAGE analysis of proteins in the experiment.

2.2 Remove GST tag and GST-tagged proteases

The procedure of removing GST tag and GST-tagged proteases is similar to the previous example of removing His tag and His-tagged proteases (section 2.1).

The following is an example of purification of GST-tagged Streptokinase C containing PreScission cleavage site and subsequent tag cleavage and removal.

Fig 3. Purification of GST-tagged Streptokinase C on GSTrap 4B 1ml column.

After purification on a GSTrap 4B column, the purified protein with GST tag was exchanged to a buffer suitable for PreScission cleavage using dialysis and treated with PreScission protease for 5 hours at room temperature.

After cleavage the solution was applied to a GSTrap 4B column again. The flow through fraction (lane 7) contains the target without GST tag.

Fig 4. Removal of tag and non-tagged proteases

Fig 5. SDS-PAGE analysis of proteins in the experiment

2.3 Remove tag and non-tagged proteases: thrombin or Factor Xa

The procedure here is similar to that with tagged proteases, except the last step in the following figure. Here we show the procedure for his-tagged proteins. After the tag cleavage in solution is completed, equilibrate a HiTrap Benzamidine FF (High Sub) column (prepacked with Benzamidine Sepharose Fast Flow) with the recommended binding buffer. Connect the HisTrap column on top of the HiTrap Benzamidine column. Load the solution to the combined columns and the flowthrough will contain the de-tagged protein.

Summary

Affinity tags greatly enhance efficiency and flexibility in protein purification. However, tags may need to be removed if they affect the protein's function, structure, or stability. This is typically done using specific proteases like thrombin or Factor Xa. Tag removal can be performed through batch or on-column cleavage, followed by the removal of cleaved tags, uncleaved proteins, and proteases to obtain pure target proteins.

On-column cleavage is an automated option that removes both the tag and protease using chromatography systems. Tagged proteases can be removed using the same affinity chromatography resin used for the target protein.

Cleavage in solution involves incubating the tagged protein with protease, followed by purification to remove the protease and cleaved tags.

We described detailed procedures for the removal of tags from GST-tagged and His-tagged proteins, including buffer compositions and incubation conditions.

Product information