I am working with a MMLV-RT (Moloney murine leukemia virus- Transcriptase Reverse). I am expressing in E. coli pTGroE and purifying in a Chelating Sepharose loaded with Ni2+. I am eluting the protein with 500 mM of Imidazole, however, there are a lot of contaminants together (gel attached). I think these contaminants are chaperones, due to this strain produces two chaperones. I read some protocols, that the autors use 0.5% Triton X-100, 10% (v/v) glycerol, together with the buffer containing imidazole. However, we do not have BCA kit to quantify protein, and Bradford is not possible to use with thi detergent and glycerol. Someone has a suggestion to improve the purification? OBS: it is not possible to see the band in the SDS-PAGE, but in the Western Blot we have a band in 56 kDa. Therefore, the protein is minority in this sample.
For a protein such as this that is expressed at a very low level, at least in the soluble phase, it is essentially impossible to purify it by a single step of immobilized metal affinity chromatography. Several chromatography steps will be needed, and even then it may not be possible to purify it to homogeneity.
The first thing to do is to find out whether the protein is mostly found in the insoluble phase, i.e. the pellet after breaking open the cells. If so, then you should work on trying to improve its expression in the soluble phase. At the same time, if the protein is found in inclusion bodies in high yield, you could consider trying to refold it. Although this is challenging, it has the advantage that the inclusion bodies, after washing, are already essentially the pure protein.
If the protein is in the soluble phase but is expressed only at a very low level, then you should start working on trying to improve its expression. You may have to try different vectors and expression strains.
You cannot even see your protein in this SDS PAGE, do you see it in other fractions than elution? (load, wash). In this case you must work with imidazole concentrations in order to elute more of your protein. How big your elution pool was?
If it is not the case and it is weakly expressed, you must work to improve the quantity produced, otherwise you cannot perform the purification steps with such a low yield.
Addition of Triton X-100 implies that the protein is not entirely soluble - have you possibly done WB with your pellet (the one you obtain after cell disruption and centrifugation)? 5-10% glycerol is usually added to stabilize the protein during purification. Since you have many other proteins in your sample, it is debatable if you can quantify your protein spectroscopically or through colorimetry - the most correct way here would be to quantify it from the WB using a known standard.
You can improve your his-tag purification using less of the Ni-NTA beads so that the weakly associating contaminants become "outcompeted" by the his-tagged MMLV-RT. However, as others have mentioned, you may need to optimize expression/solubility first.
Good luck!
Thanks a lot for all the answers. Yes, I am planning some experiments to improve the yield of this protein, is indeed very low. I am using Western Blot to quantify the protein and I am also using amicons to concentrate the sample before the Western. I tested the pellet and I could not find the protein there. I tested different strains of E. coli: E. coli BL21, E. coli BL21 RIL e E. coli pTGroE. I am thinking also to change the vector. Thanks a lot for you help!
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