My N -terminal GST tagged protein fail to purify using this condition: 30ml sample (3mg of crude protein) injected at flowrate (0.5ml/min), 40ml wash (PBS pH 7.3) and 5ml elution (50mM Tris HCl + 10mM reduced glutathione, pH 8). For washing and elution, the flowrate was 1ml/min. Buffers were prepared all according to the manual. For your information, based on SDS -PAGE analysis, the crude has decent amount of GST tagged protein. Therefore, I suspected that the GST tag was hidden in the conformation. The protein structure was not discovered yet so I can only hypothesized. Based on previous structural analysis, the N -terminal was predicted to be at the cytoplasmic region. Does this means, whatever tag that I put in the N -terminal will be forever hidden in the structure? Should I cleave the tag and purify the protein using other means (IEX/ SEC/ HIC)?
Since this is an integral membrane protein, it will require a detergent to get it into solution and keep it in solution during the purification. Since you did not mention using any detergent in your procedure, I suspect the protein was not in solution and therefore did not bind to the column.
Adam B Shapiro thank you so much for your answer :DD
I have a few additional question,
1. If the protein is not in solution, where does it reside?
2. What kind of detergent should I add the best? I plan to add 0.1% Triton X- 100 as per the manual into the elution buffer, should I also add some into the lysis buffer?
3. The resin manual mentioned that nonspecific hydrophobic interactions with the medium cause protein aggregation, preventing solubilization and elution of tagged proteins, hence recommending the addition of 0.1% Triton. Should I clear up the potential aggregation before another purification trial
Thank you!!
Membrane proteins are more difficult to purify than soluble proteins and a detergent or other strategy is likely necessary. In addition, it may be worth running some tests on your tag if you haven't already. When purifying a new protein, it's common to have to move the tag around (N-term, C-term, loops) to find a position that works and there may or may not be an apparent reason for a given location not working. If you have the resources, a small-scale immunoprecipitation reaction (in a suitable detergent lysis buffer) could be used to determine if the tag is accessible.
The first question is whether the protein is being expressed in the membrane. If not, it will be in the form of an insoluble material. When you lyse the cells, if you do not centrifuge the lysate the insoluble material will be floating around in the lysate. If you apply the lysate to a column, the insoluble material will either pass through without binding, or get stuck in the column and clog it up. If you centrifuge the lysate first, the insoluble material will be in the pellet.
It may be possible to recover the insoluble protein from the pellet by dissolving the pellet in a detergent. Nonionic (such as Triton X-100, but there are many others) and zwitterionic detergents are usually used for purifying membrane proteins when it is necessary to maintain the native folded structure of the protein, although it is unlikely that the transmembrane protein is in its native state in the pellet. The GST portion may have the native structure, so you may still be able to use that for affinity purification if you can get the protein into solution with a non-denaturing detergent.
Once you have purified the protein, you can either keep it in solution in detergent micelles, or reconstitute it into lipid bilayer membranes or nanodiscs.
- Badges
- Science topic
- Similar topics
- Human-Computer Interaction
- Collaboration