Removal of the GST tag is often necessary to be able to perform functional or structural studies of the target protein. Tagged proteins containing a PreScission Protease, thrombin, or Factor Xa recognition site can be cleaved either while bound to Glutathione Sepharose® or in solution after elution. Cleavage releases the target protein from the column and allows elution using the binding buffer. The GST moiety remains bound to the medium.
PreScission Protease itself has a GST tag and therefore will bind to Glutathione Sepharose; it will thus not co-elute and contaminate the cleaved target protein. Cleavage with PreScission Protease is very specific, and maximum cleavage is obtained in the cold (the protein is most active at 4 °C), thus improving the stability of the target protein.
If thrombin or Factor Xa are used for cleavage of the tag, a convenient way to remove these enzymes is to connect in series one GSTrap™ FF column and one HiTrap™ Benzamidine FF (high sub) column. During the elution the cleaved product passes directly from the GSTrap™ into the HiTrap™ Benzamidine FF (high sub). The cleaved target protein passes through the HiTrap™ Benzamidine FF (high sub) column but the proteases bind. Thus in a single step the enzymes are removed and a pure cleaved target protein is achieved (Figure 5.17). Note, however, that thrombin and Factor Xa may produce a less specific cleavage than PreScission Protease and that sometimes the target protein can be fragmented itself.
1 PreScission Protease is a tagged protein of glutathione S-transferase and human rhinovirus type 14 3C protease.
The amount of enzyme, temperature, and length of incubation required for complete digestion varies according to the specific GST-tagged protein produced. Optimal conditions should always be determined in pilot experiments.
If protease inhibitors (Table 5.6) have been used in the lysis solution, they must be removed prior to cleavage with PreScission Protease, thrombin, or Factor Xa. (The inhibitors will usually be eluted in the flowthrough when sample is loaded onto a GSTrap™ column.)
Cleavage of tagged proteins is most commonly performed on milligram quantities of tagged protein suitable for purification on GSTrap™ columns. Protocols that follow describe manual cleavage and purification using a syringe and a 1 mL or 5 mL GSTrap™ column. The protocols can be adapted for use with GST MultiTrap™ or GST SpinTrap™ columns to work at smaller scales.
For quick scale-up of purifications, two or three GSTrap™ columns can be connected in series (back pressure will be higher). Further scaling-up is possible using GSTPrep™ FF 16/10 columns or columns packed by the user. Protocols below are included for column or batch format using Glutathione Sepharose® 4 Fast Flow, but this medium can easily be replaced with Glutathione Sepharose® High Performance or Glutathione Sepharose® 4B depending on what is the preferred chromatography medium in the lab.
Figure 5.17Cleavage of GST Tag using Thrombin or Factor Xa.
Cleavage and purification of GST-tagged protein bound to GSTrap™ FF |
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The protocol below is an example optimized for 8 mg of target protein. It is worth estimating how much target protein is applied to the column, as this allows one to minimize the amount of protease added.
The incubation times are starting points and may need to be changed for an optimal yield of cleaved target protein.
For PreScission Protease: The eluate will contain the protein of interest, while the GST moiety of the tagged protein and the PreScission Protease (also GST-tagged) will remain bound to the GSTrap™ column. This means that the protein of interest will not be contaminated with protease and thus no additional purification will be required to purify the target protein from the protease.
For thrombin and Factor Xa: The eluate will contain the protein of interest and thrombin or Factor Xa, respectively, while the GST moiety of the tagged protein will remain bound to the GSTrap™ column. Thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap™ Benzamidine FF (high sub) column in series after the GSTrap™ column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap™ column onto the HiTrap™ Benzamidine FF (high sub) column. This second column captures the thrombin or Factor Xa, thus enabling the collection of free protein in the eluent. Refer to Figure 5.20 for an example of the purification and on-column cleavage of GST-tagged SH2 domain using thrombin and GSTrap™ FF, with sample cleanup accomplished using HiTrap™ Benzamidine FF (high sub) column in series with GSTrap™ FF. Removal of thrombin and Factor Xa using HiTrap™ Benzamidine FF (high sub) later on in this chapter for the procedure.
Appendix 2 (Characteristics of Glutathione Sepharose® products) for details on regenerating the GSTrap™ column for subsequent purifications.
1. Purification of human hippocalcin using GSTrap™ FF columns in series with on-column cleavage by PreScission Protease
The gene for human hippocalcin, a member of the neuron-specific calcium-binding protein family, was cloned into a pGEX vector containing a PreScission Protease site adjacent to the GST tag. The expressed tagged protein was captured on a GSTrap™ FF 1 mL column. The column was then incubated overnight at 4 °C and for an additional 2 h at room temperature with PreScission Protease (which is GST-tagged itself). Following on-column cleavage, a second GSTrap™ FF 1 mL column was placed in series after the first to remove any PreScission Protease, uncleaved GST-tagged protein, or free GST tag that could co-elute with the sample during the additional wash with binding buffer (Figure 5.18). For every gram of wet E. coli cells, 10 mg of pure, untagged hippocalcin was obtained.
Figure 5.18.Purification of human hippocalcin-GST-tagged protein with on-column cleavage and post-cleavage removal of PreScission Protease using GSTrap™ FF columns. (A) Chromatogram showing purification of hippocalcin. (B) SDS-PAGE analysis of various sample processing steps. ExcelGel SDS Gradient, 8–18, Coomassie blue staining.
2. Automatic removal of the GST tag with PreScission Protease
This example of automated tag removal uses ÄKTAxpress. All multistep purification protocols in ÄKTAxpress can be combined with automated on-column tag cleavage. Tag cleavage is always performed on the affinity column prior to further purification steps. When the cleaved protein has been eluted, the affinity column is regenerated and affinity tag, tagged protease, and remaining uncleaved protein are collected in a separate outlet. The procedure involves binding the tagged protein, injection of protease, incubation, elution of cleaved protein, and collection in capillary loop(s), followed by further purification steps.
The example in Figure 5.19 shows purification results for a GST-tagged protein, GST-purα (Mr 61 600), expressed in E. coli. The Mr of the cleaved product is 35 200. After harvest, cell lysis was performed by sonication. The samples were clarified by centrifugation prior to sample loading.
AC and SEC were performed on ÄKTAxpress using columns as indicated in the figure. The purity of each sample was analyzed by SDS-PAGE (Coomassie staining). The reduced samples were applied on an ExcelGel SDS-polyacrylamide gel.
Figure 5.19.(A) Two-step protocol for automatic GST-tagged protein cleavage with PreScission Protease and purification. (B) Analysis by SDS-polyacrylamide gel (Coomassie staining) of the untagged target protein after purification and cleavage.
3. Purification and on-column cleavage of GST-tagged SH2 domain using thrombin and GSTrap™ FF. Direct removal of thrombin using HiTrap™ Benzamidine FF (high sub) column in series with GSTrap™ FF
The following application describes the purification of GST-SH2 (Mr 37 000) on a GSTrap™ FF 1 mL column, followed by on-column cleavage with thrombin (Figure 5.20). After the thrombin incubation step, a HiTrap™ Benzamidine FF (high sub) 1 mL column was placed in series after the GSTrap™ FF column. As the columns were washed with binding buffer and later with high-salt buffer, the cleaved SH2-tagged protein and thrombin were washed from the GSTrap™ FF column onto the HiTrap™ Benzamidine FF (high sub) column. Thrombin was captured by this second column, thus enabling the collection of pure thrombin-free untagged target protein in the eluent (Figure 5.20A). Complete removal of thrombin was verified using the chromogenic substrate S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma) for detection of thrombin activity (Fig 5.20B). This entire procedure could be completed in less than one day.
Figure 5.20.Purification of GST-SH2 GST-tagged protein with on-column cleavage and post-cleavage removal of thrombin using GSTrap™ FF and HiTrap™ Benzamidine FF (high sub) columns. (A) SDS-PAGE analysis of various sample processing steps. ExcelGel SDS Gradient 8–18, Coomassie blue staining. (B) Chromatogram (blue: absorbance at 280 nm) and thrombin activity curve (red) demonstrating all steps in the purification of the SH2 domain.
4. On-column cleavage of a GST-tagged protein using thrombin on a GSTrap™ FF column
To demonstrate the efficiency of on-column cleavage in conjunction with purification, a GST-tagged protein containing the recognition sequence for thrombin, was applied to GSTrap™ FF 1 mL. After washing, the column was filled by syringe with 1 mL of thrombin solution (20 U/mL in PBS, pH 7.3) and sealed using the supplied connectors. After incubation for 16 h at room temperature, the target protein minus the GST moiety was eluted using PBS, pH 7.3, and the bound GST was subsequently eluted using elution buffer (Figure 5.21). The cleavage reaction yield was 100%. Intact GST-tagged protein was not detected in the eluate by SDS-PAGE and silver staining (Figure 5.21C, lane 5).
Figure 5.21.On-column thrombin cleavage of a GST-tagged protein. (A) Equilibration, sample application, and washing of a GST-tagged protein on GSTrap™ FF 1 mL were performed using ÄKTA chromatography system. After washing, the column was filled by syringe with 1 mL of thrombin (20 U/mL) and incubated for 16 h at room temperature. (B) GST-free target protein was eluted using PBS, pH 7.3. GST was eluted using 10 mM reduced glutathione. (C) SDS-PAGE followed by silver staining. The GST-free target protein fraction also contained a small amount of thrombin not detectable by SDS-PAGE (lane 6). The thrombin can be removed using a HiTrap™ Benzamidine FF (high sub) column.
Purification and Cleavage
The protocol below is an example optimized for 8 mg of target protein. It is worth estimating how much target protein is applied to the column, as this allows one to minimize the amount of protease added.
The incubation times are starting points and may need to be changed for an optimal yield of cleaved target protein.
For PreScission Protease: The flowthrough will contain the protein of interest, while the GST moiety of the tagged protein and the PreScission Protease will remain bound to the GSTrap column. This means that the protein of interest will not be contaminated with protease and thus no additional purification will be required to purify the target protein from the protease.
For thrombin and Factor Xa: The flowthrough will contain the protein of interest and thrombin or Factor Xa, respectively, while the GST moiety of the tagged protein will remain bound to the GSTrap column. The thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap Benzamidine FF (high sub) column in series after the GSTrap column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap column onto the HiTrap Benzamidine FF (high sub) column. This second column captures the thrombin or Factor Xa, thus enabling the collection of pure protease-free protein in the eluent. Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub) for the procedure.
Appendix 2 (Characteristics of Glutathione Sepharose products) for details on regenerating the GSTrap column for subsequent purifications.
Cleavage and purification of GST-tagged protein bound to Glutathione Sepharose in batch mode
Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B can all be used for cleavage and purification of GST-tagged proteins in batch.
Recommended Buffers |
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Glutathione Sepharose media are supplied in 20% ethanol. The media are used at a final slurry concentration of 50%.
Glutathione Sepharose must be thoroughly washed with PBS to remove the ethanol storage solution because residual ethanol may interfere with subsequent procedures.
Assume 8 mg of GST-tagged protein bound per mL of chromatography medium.
The incubation times in steps 4a and 4b are starting points and may need to be changed for an optimal yield of cleaved target protein.
For PreScission Protease: The eluate will contain the protein of interest, while the GST moiety of the tagged protein and the PreScission Protease will remain bound to the Glutathione Sepharose. This means that the protein of interest will not be contaminated with protease and thus no additional purification will be required to purify the target protein from the protease.
For thrombin and Factor Xa: The eluate will contain the protein of interest and thrombin or Factor Xa, respectively, while the GST moiety of the tagged protein will remain bound to the Glutathione Sepharose. The thrombin or Factor Xa can be removed from the protein of interest using HiTrap Benzamidine FF (high sub). This column captures the thrombin or Factor Xa, thus enabling the collection of pure protease-free protein in the eluent. procedure below.
Removal of thrombin and Factor Xa using HiTrap Benzamidine FF (high sub)
Binding buffer:
0.05 M Tris-HCl, 0.5 M NaCl, pH 7.4
Elution buffer alternatives for eluting the protease:
0.05 M glycine-HCl, pH 3.0
10 mM HCl, 0.5 M NaCl, pH 2.0
20 mM p-aminobenzamidine in binding buffer (competitive elution) 8 M urea or 6 M Gua-HCl (denaturing solutions)
Recommended flow rates are 1 mL/min (1 mL column) or 5 mL/min (5 mL column).
Thrombin activity can be followed by taking aliquots of the fractions and measuring at 405 nm using S-2238 (Chromogenix, Haemochrom Diagnostica AB; supplier in US is DiaPharma) as substrate.
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