I am purifying a protein expressed in pLysS cells. The construct is in pET28 vector and having His tag with TEV cleavage site . Now when I ran the SDS-PAGE gels after TEV cleavage and purification, I could find double bands, a band at the size of the protein of interest and another at slightly higher size(around 1-2kD). I even performed FPLC and could find single peak but when I ran the gel I could find the double bands. What could be reason for the same and how to rectify this?
The probable answer could be:
1)the occurance of isozyme form of your protein.
2) reading through the stop codon of your vector which can be fixed by adding tandom stop codon in your vector pET28
Hi there,
The full length protein might be partially cleaved at 1-2kD distance from N or Cter during expression/purification process. If the 2 polypeptide resulting from the cleavage remain associated in the native state of the protein (disulphide bond or even non covalent interaction) then it explains why a single peak during SEC gives 2 bands (in fact 3 bands but the 1-2kD band might not be visible on gel) in denaturing condition. If it is so then you have to find the factor responsible for the cleavage: TEV or cellular protease. Is the doublet only noticable after TEV or does it occur from the start? If the former, TEV might be cleaving partially a second site, if the latter the use of protease inhibitor should improve the homogeneity of the purified product.
You should also try to identify the content of the 2 band by isolating each band and perform MS fingerprint and so identify the difference between the 2 bands.
Ridhima Wadhwa Thanks for the suggestion. I don't think their should be a problem of isozyme as the protein domain is cloned in a bacterial vector.
Dominique Liger Thanks for the suggestion. Well I could see the doublets even before TEV cleavage but yes its more like a broad band whereas after the TEV cleavage the doublets can be distinguished.
Post-translational modification of proteins can also cause doublets that are very close together in SDS-PAGE, but inseparable by gel filtration or ion exchange chromatography. I had this once with Hsc70 (Article Binding of ATP and ATP analogues to the uncoating ATPase Hsc...
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Engelbert Buxbaum expression system is bacterial so I don't think such problem should occur
I think you should provide more detail if you want useful answers.
1-2 kDa corresponds to the his-tag plus TEV recognition sequence. So most likely it is incomplete clevage. You "performed FPLC" - what technique did you use? ion exchange? size exclusion? Using which column? Depending on the size of your protein you may or may not see separation on SEC. 1 kDa difference is not a lot on a 30 kDa protein. It may only be a small hint of a shoulder on the peak that is very difficult to see. And there is probably little difference in charge so you get little or no separation on ion exchange - depending on the resolution of the column you use.
A little more information will enable us to help you better. What size is your protein? What techniques did you use for the purification? etc
Shubhashish Chakraborty : Regulatory protein modification occurs in prokaryotes just as in eukaryotes.
I agree with Buxbaum, post translation modifications do occur in bacterial cells. I work with a kinase that is degraded when phosphorylated in bacterial cells.
What Pontoppidan said makes a lot of sense too. Maybe your not getting full cleavage and you need to optimize.
Bo Pontoppidan I am using size exclusion chromatography and the protein size is 7kD.
Engelbert Buxbaum Tracess Smalley well thanks for the information. I am too suspecting about incomplete cleavage. I initially treated TEV for an hour, then with the same concentration kept for 3 hours and then overnight. In all three cases I could see the doublets.
About post translational modification I think I have to read more.
if it is incomplete cleavage than you should only be able to bind one of the two proteins to a second IMAC column.
if it is modification, you should see doublets even before purification/cleavage and this situation should not change upon repurification of the cleavage products.
you may even use a Western blot to see if both proteins have the his-tag
Good luck
Jürgen
Shubhashish Chakraborty What are your SDS-PAGE assay conditions? And are there any di-sulphides in your protein? It could be an artefact of the SDS-PAGE assay rather than a feature of your protein/process.
I find that using SDS sample buffer and heating to 95°C (5 min) for denaturation causes di-sulphides to shuffle in my protein and therefore give me doublets in my non-reducing gel.
I switched to LDS buffer and heating to 70°C (10 min) prevented the doublet from forming but still gave sufficient denaturation of the protein.
It is most probably a uncomplete cleavege. It can also caused by extra detergent in the buffers (like triton-X ). This can interfere with the homogeneity of SDS cover of the heat denatured protein.
I have checked both the bands using mass spec. One of the band is a cleaved N terminal portion of my protein of interest.
Peter Kysel I am using normal Tris-NaCl buffer pH 7.5, no detergent
James Cummins There are only two cysteine residues in my protein and I don't think their is any disulphide bond
What is the size of the band corresponding to the cleaved N' portion?
You mentioned that you observe two bands in the gel. One is slightly higher in size than the protein of your interest which I believe you meant was the smaller band. So, if the larger band is the full length protein then what is its size as seen in the gel and the theoretical mass?
yes, I have the same doubt as Dr. Shubham; What were the molecular weights of the intended protein (expressed protein) and the fragmented N terminal? It would help us gain knowledge.
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