HiTrap^® Benzamidine FF for Thrombin & Factor Xa Removal

To protect the fusion protein from proteolytic degradation prior to enzymatic cleavage with PreScission Protease, thrombin, or Factor Xa, it may be necessary to remove proteases from the sample. Additionally, following enzymatic cleavage, it may be necessary to remove thrombin or Factor Xa from the sample. Benzamidine Sepharose 4 Fast Flow (high sub) provides a convenient and highly specific medium for the removal of trypsin and trypsin-like serine proteases, not only from enzymatic digests but also from cell culture supernatants, bacterial lysates, or serum.

Benzamidine Sepharose 4 Fast Flow is available in either prepacked 1 mL or 5 mL HiTrap^® columns or in packages for scaling up purifications. HiTrap^® columns can be operated with a syringe together with the supplied adapters, a pump, or a liquid chromatography system such as ÄKTA. See Table 1 for a selection guide of purification options.

Characteristics of HiTrap^® Benzamidine FF (high sub) are summarized in Appendix 5 (Characteristics of Glutathione Sepharose and HiTrap^® Benzamidine FF (High Sub) Media and Columns).

Table 1. Selection guide for purification options to remove thrombin and Factor Xa.

Column (prepacked) or medium	Binding capacity for trypsin	Comments
HiTrap® Benzamidine FF (high sub), 1 mL	> 35 mg trypsin	Prepacked 1 mL column
HiTrap® Benzamidine FF (high sub), 5 mL	> 175 mg trypsin	Prepacked 5 mL column
Benzamidine Sepharose 4 Fast Flow (high sub)	> 35 mg trypsin/mL medium	For column packing and scale-up

Reagents Required

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 guanidine-HCl (Gua-HCl) (denaturing solutions)

Recommended flow rates are 1 mL/min (1 mL column) or 5 mL/min (5 mL column).

1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied), laboratory pump, or chromatographic system “drop to drop” to avoid introducing air into the column.
   
   
2. Remove the snap-off end.
   
   
3. Wash the column with 5 column volumes of distilled water to remove the storage buffer (0.05 M acetate buffer, pH 4, containing 20% ethanol).
   
   
4. Equilibrate the column with 5 column volumes of binding buffer.
   
   
5. Apply the sample using a syringe fitted to the Luer connector or by pumping it onto the column. Recommended flow rates for sample application are 1 mL/min for 1 mL column and 5 mL/min for 5 mL column. Collect the flowthrough and reserve. It contains the protease-depleted material to be saved. Apply a small volume of extra binding buffer to collect all desired material from the column.
   
   
6. Wash the column with 5 to 10 column volumes of binding buffer, collecting fractions (0.5 to 1 mL fractions for 1 mL column and 1 to 3 mL fractions for 5 mL column) until no material appears in the effluent (monitored by UV absorption at 280 nm).
   
   
7. Pool fractions from flowthrough and/or wash that contain the thrombin- or Factor Xa-free material (monitored by UV absorption at 280 nm).
   
   
8. For reuse of column, elute the bound protease with 5 to 10 column volumes of the elution buffer of choice. If the eluted thrombin or Factor Xa is to be retained for reuse, buffer exchange the fractions containing the protease using a desalting column. If a low pH elution buffer has been used, collect fractions in neutralization buffer.
   
   
9. After all protease has been eluted, wash the column with binding buffer so it is ready for reuse.
   
   
10. For longer-term storage, store in a buffer containing 20% ethanol in 0.05 M acetate buffer, pH 4, at 4 °C to 8 °C.

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.

Application Example

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 (M_r 37 000) on a GSTrap™ FF 1 mL column, followed by on-column cleavage with thrombin (Figure 5.7). 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 1A). 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 (Figure 1B). This entire procedure could be completed in less than one day.

Figure 1. 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.