Experimental purification of fusion proteins by glutathione agarose affinity chromatography
Experimental purification of fusion proteins by glutathione agarose affinity chromatography
The exogenous protein expressed in the pGEX vector is fused to glutathione S transferase and can therefore be purified by glutathione-agarose affinity chromatography. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experimental purification of fusion proteins by glutathione agarose affinity chromatography
Materials and Instruments
E. coli cells expressing GST fusion proteins Move makings For more product details, please visit Aladdin Scientific website.
Dithiothreitol Glutathione elution buffer Phosphate buffered sodium salt solution TritonX-100 DNase Lysozyme RNase Thrombin, enterokinase, or Xa factor solution SDS-polyacrylamide gel
SorvallSS-34 Turning head or equivalent Glutathione-agarose resin Hypodermic needle
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Dithiothreitol (1 mol/L)
Glutathione elution buffer
10mmol/L Reduced Glutathione
50 mmol/L Tris-Cl (pH 8.0)
Phosphate buffered sodium salt solution (PBS) (4°C)
TritonX-100 (0.2%V/V)
Enzymes and buffers
DNase (5 mg/ml)
Lysozyme
RNase(5 mg/ml)
Thrombin, enterokinase, or Factor Xa solution
Refer to manufacturer's manual for preparation and storage of solutions.
Gel
SDS-Polyacrylamide Gel (10%)
Refer to Appendix 8 for preparation of SDS-polyacrylamide gels for protein isolation.
Centrifuge and rotor head
SorvallSS-34 turntable or equivalent
Special equipment
Glutathione-Agarose Resin
Mix resin to 50% homogenate with 20% ethanol if necessary.
Hypodermic needles (18# 22# and 25#)
Carrier and Bacterial Strain
E. coli cells expressing GST fusion proteins (cell precipitates prepared in step 17 of Scheme 1 or 2).
Methods
Treatment with glutathione-agarose resin
1. Gently invert the container containing the glutathione-agarose resin to mix the resin into a homogenate.
2. Place a portion of the homogenate into a 15 ml polypropylene tube (2 ml of homogenate per 100 ml of bacterial culture).
3. Centrifuge at 500 g (2100 r/min Sorvall SS-34 turntable) at 4°C for 5 min and carefully remove the supernatant.
4. Add 10 times the volume of cold PBS to the resin, invert several times, mix well, and centrifuge at 4°C for 5 min at 500 g (2100 r/min Sorvall SS-34 head), removing the supernatant carefully.
5. Add lml of cold PBS per ml of resin to make a 50% homogenate, invert several times, mix well, and place suspension on ice until ready for use.
Preparation of cell extracts
6. For every 100 ml of culture, suspend the cell precipitate (Scheme 1, step 17) in 4 ml of PBS.
7. Add lysozyme to a final concentration of 1 mg/ml and leave on ice for 30 min.
In some protocols, cells are lysed by sonication or Fuchsin crushing prior to affinity purification of the GST fusion protein. Methods using lysozyme are more reproducible and do not cause catastrophic cell lysis. The latter releases outer membrane proteins and other molecules that can cause aggregation of GST fusion proteins or that can be co-purified with them.
8. 10 ml of 0.2% TritonX-100 is forcefully injected into the viscous cell lysate with a syringe and mixed by shaking vigorously several times.
Add DNase and RNase to a final concentration of 5ug/ml and incubate with shaking at 4°C for 10 min. Centrifuge at 3000 g (5000r/min Sorvall SS-34 head) for 30 min to remove insoluble cellular debris. The supernatant (cell lysate) was transferred to a new tube and DTT was added to a final concentration of l mmol/L.
To prevent the resin from getting clogged during purification, the supernatant should be filtered with 0.45um membrane.
The purpose of adding TritonX-100 is to prevent protein aggregation. Denaturants that can solubilize fusion proteins without interfering with glutathione-agarose affinity purification include 1% (V/V) TritonX-lOO, 1% (WV) Tween-20 containing 10 mmol/L DTT, 0.03% (m/V) SDS, and 1.5% (m/V) dodecahydrexyl sarcosine (Smith and Johnson 1988; Frankel et al. 1991; Frankel et al. 1991; Frankel et al. 1991; Frankel et al. 1991; Frankel et al. 1991). Frankel et al.1991; Grieco et al,1992). The addition of Tween-20, SDS, and N-dodecyl sarcosine can be used in place of or in conjunction with TritonX-100. See Troubleshooting: insoluble proteins.
Purification of fusion proteins
9. Mix cell lysates with an appropriate amount of 50% glutathione-agarose resin homogenate, add 2 ml of resin per 100 ml of bacterial culture and shake gently at room temperature for 30 min.
10. The mixture is centrifuged at 500 g (2100 r/min SorvallSS-34 head) at 4°C for 5 min, the supernatant is carefully removed and a small sample is retained for SDS polyacrylamide gel electrophoresis.
11. 10 times the volume of the column bed is added to the precipitate, and the tube is mixed several times by inverting the centrifuge tube to wash away proteins that are not bound to the resin.
12. Centrifuge at 500 g (2100 r/min SorvallSS-34 head) for 5 min at 4°C, carefully remove the supernatant and leave a small sample for SDS polyacrylamide gel electrophoresis.
13. Repeat steps 11 and 12 twice
14. Bound GST fusion proteins can be eluted with glutathione elution buffer or cleaved by thrombin, enterokinase or Xa factor to release the target protein.
Elution of fusion proteins with glutathione
a. Add 1x the column bed volume of Glutathione Elution Buffer to the precipitate and elute the bound proteins from the resin by gentle agitation for 10 min at room temperature.
b. Centrifuge as in step 12 and transfer the supernatant (containing the eluted fusion protein) to a new tube.
c. Repeat steps a and b twice, combining the supernatants three times.
After the above elution steps, some proteins may still be bound to the resin in significant amounts and may require further elution. SDS polyacrylamide gel electrophoresis can be used to monitor the amount of GST fusion proteins in the eluate. For GST fusion proteins, typically 1A280=0.5 mg/ml, the 280mn absorption can be measured to estimate the yield of the fusion protein, or the standard colorimetric method (Lowry or Bradford) can be used to determine the yield of the fusion protein, but in the case of the Lowry method, the sample must be dialyzed with 2000 times the volume of 1XPBS to remove the glutathione. If the Lowry method is used, the sample must first be dialyzed in 2000x volume of 1XPBS to remove glutathione, which can interfere with the assay, while the Bradford method is not affected by glutathione.
Recovery of target protein from bound GST fusion protein by proteolytic digestion
a. Add thrombin, enterokinase, or Factor Xa (depending on the site in the fusion protein) to the resin bound to the fusion protein. Add 50 units of protease dissolved in lml PBS per ml of resin. The tubes are inverted several times to mix and shaken at room temperature for 2 to 16 h. The exact time is determined by small-scale experiments.
b. Centrifuge at 500 g (2100 r/min Sorvall SS-34 head) for 5 min at 4°C and carefully transfer the supernatant to a new tube.
The GST is still bound to the resin, while the target protein is in the supernatant, as is the protease. Separate white and protease by conventional chromatography or SDS polyacrylamide gel electrophoresis.
15.10% SDS polyacrylamide gel electrophoresis analyzes the protein composition of the sample at each step (cell extraction, washing, and elution). 