Key assays in enzymology for biochemical characterization of proteins in vitro require high concentrations of the purified protein of interest. Protein purification protocols must combine efficiency, simplicity, and cost-effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on an N-terminal glutathione sepharose (GST) tag and a C-terminal 10xHis tag, both fused to the protein of GST. interest. The latter construct is used to generate baculoviruses, for infection of Sf9-infected cells for protein expression.
GST is a fairly long tag (29 kDa) that serves to ensure the efficiency of the purification. However, it could influence the physiological properties of the protein. Therefore, it is subsequently cleaved from the protein using the PreScission enzyme. To ensure maximum purity and remove cleaved N-terminal 10xHis-GST-tagged Recombinant, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique relies on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and thus enriches full-length proteins.
The method presented here does not require an expensive instrument setup, such as FPLC. In addition, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to remove contaminating nucleic acids. In summary, the combination of two different flanking tags at the N- and C-terminus and the ability to cleave one of the tags ensures the recovery of a full-length, highly purified protein of interest.
Keywords: glutathione S-transferase (GST), pGEX, protein expression, protein purification, thrombin, factor Xa, fusion tags
Affinity purification of GST fusion protein
Soluble GST fusion proteins are easily purified using an immobilized glutathione sepharose column. There are several immobilized glutathione chromatography media options available to purify soluble GST fusion proteins from bacterial cell lysates. The protocol described below is adapted from the manufacturer’s recommendation using Glutathione Sepharose 4B poured onto a column and using a peristaltic pump to control flow rates. Protease inhibitors and reducing agents should be added to the buffers as required to minimize proteolysis of the fusion protein. One exception is that serine protease inhibitors must be removed from the glutathione buffer prior to enzymatic removal of the GST moiety, as they will inhibit enzyme activity.
Save a small aliquot from each purification step for SDS-PAGE analysis to monitor fusion protein location throughout the purification. A given column or batch of resin should be used exclusively with a single fusion protein to minimize potential cross-contamination. As an alternative to column purification, a protocol describing batch purifications. Batch purifications are quick and easy, but often the yield and purity of the protein obtained will be somewhat less than that obtained by chromatographic separations. To minimize proteolysis, all protein purification steps should be carried out at 4°C, unless otherwise indicated.
- Resuspend and pour 20 mL of Glutathione Sepharose 4B resin into a 2.5 × 8 cm column.
- Thoroughly wash Glutathione Sepharose with 5–10 bed volume PBS at 1.5 mL/min to remove ethanol stock solution.
- Resuspend pelleted E. coli cells in 15 ml cold Lysis buffer (cells can be fresh or thawed frozen cell pellets).
- Lyse cells by sonication on ice (~10 times for 10 seconds each with 1-minute rest between bursts to minimize heating of the sample). Save 50 μl of lysate for on-gel analysis and transfer the rest to a 60 ml centrifuge tube.
- Centrifuge the lysate at 48,000 × g for 20 min at 4 °C.
- Decant the supernatant into a clean 50 mL centrifuge tube.
- Resuspend the pellet in 15 ml PBS buffer using a Dounce homogenizer.
- Run 5-10 μl of the lysate, supernatant, and resuspended pellet on an SDS-PAGE gel to verify that the fusion protein is in the supernatant fraction. If the fusion protein is in the pellet fraction, for tips on improving soluble protein expression for methods of extracting protein from inclusion bodies.
- Load the soluble fusion protein onto the equilibrated glutathione sepharose column using a flow rate of 0.1 mL/min. Collect fractions and run gels to verify fusion protein binds to column and capacity has not been exceeded. If the fusion protein binds poorly to the resin, for various possible remedies.
- Wash the column with 5–10 bed volumes of PBS/EDTA/PMSF using a flow rate of 1.5 mL/min.
- Wash the column with 10-bed volumes of PBS/EDTA using a flow rate of 1.5 mL/min.
- Elute the fusion protein with glutathione buffer using a flow rate of 0.3 mL/min. Fractions can be monitored using A280 and SDS-PAGE analysis. Pool fractions containing the GST fusion protein. The protein can be stored at 4°C and should be ~90% pure at this stage. If problems are found eluting the fusion protein. If high levels of contamination are present, for troubleshooting tips.
Enzymatic cleavage to remove the GST affinity tag
Depending on the vector chosen, the GST affinity tag can be removed with thrombin, factor Xa, or PreScission protease, either in solution or while still bound to the column matrix. Solution cleavage offers the advantage of greater control over optimizing cleavage conditions, such as temperature, enzyme-to-substrate ratio, incubation duration, and buffer conditions. An advantage of column cleavage is the high level of purity obtained, but this comes at the expense of generally low yield due to less efficient cleavage by proteases and less control of digestion conditions.
Digestion can be performed in the glutathione buffer used to elute the protein from the affinity matrix as long as there are no serine protease inhibitors in this buffer. After incubation, the enzyme can be inhibited using a variety of protease inhibitors or removed using a HiTrap Benzamidine column. Separation of the target protein and the GST moiety can be achieved by further chromatography on the glutathione Sepharose column (after dialysis in PBS buffer) to remove GST and any uncleaved fusion protein. For information on the digestion of GST fusion proteins while bound to column matrix (use with PreScission Protease is recommended).
- Add the appropriate amount of thrombin or factor Xa to the affinity purified fusion protein and incubate at 37°C (thrombin) or 25°C (factor Xa) for the desired time.
- Inactivate the enzyme by adding 0.3 mM PMSF (final concentration) to the sample. To ensure complete inhibition, incubate the sample for 15 min at 37°C for thrombin or 30 min at 25°C for factor Xa.
- Dialyze the sample against PBS/EDTA/PMSF twice using 2 L per dialysis for a minimum of 4 h for each dialysis.
- Centrifuge the dialyzed sample for 20 min at 4,000 × g to remove any precipitated material that may have formed during the digestion or dialysis steps. At this point, the sample can be reapplied to the glutathione Sepharose column to remove the remaining GST and any undigested fusion proteins.