My protein (16 kD) is tagged with Maltose binding protein ( 42.5 kD) at N terminal and Histidine tag( ~1 kD) at C terminal. When I am expressing my protein in Rosetta Gami pLysS and BL21DE3, after running a gel I am getting a band at ~58 kD and also at ~ 17 kD.
Even after purification with Ni-NTA column, the band at 17 kD is present ( although faint) together with a band at 58 kD and ~120 kD( since protein has a tendency to form dimer). So, can it be possible that a protein during expression degrades in such a way that it naturally cleaves the tag attached to it ?
Hi there,
It is a possibility. If you have antiHis antibody, run a WB to get sure of it. If you want to avoid this likely cleavage , add protease inhibitor cocktail to your lysis buffer.
Btw what good would you get by knowing weather its the free tag or a contaminating protein which binds to your protein non specifically. Ideally as long as I am able to purify my protein in sufficient quantities I shall be happy with it by immediately following it up with gel filtration chromatography (you might like to do some other type of chromatography in case you are aware of other properties of your protein).
If you have self cleavage (does happen) you will see MBP as a major band (42 kDa) it should be stronger than your 17kDa band. If you see the 17kDa band in your lysat and not the free MBP, then it is not your protein.
Protein processing is possible, if you have a protease susceptible sequence (usually by basic residues) at the junction between MBP and your protein of interest.
You can check it with western blotting or by mass-spec if the bands are of your protein and introduce mutations at the junction to abolish protease activity.
This way you can rule out the anomalous bands.
Also, how did you see a 120 kd band on SDS-PAGE, does your protein form covalent dimer??
If your protein forms a dimer and what you are seeing is cleavage of the MBP then you should see a ~75kD band too, which would be a dimer of the cleaved and uncleaved (17+58 = 75).
If you don't see a 75kD band then it would be unlikely that the 17kD band you are seeing is the product of cleavage of the MBP from your target protein. It's simply something else that is non-specifically bound to the Ni-NTA column.
Yes Mr. Santosh C M Kumar, the protein has a tendency to for a disulphide linkage between cysteine residues, thus leading to a formation of dimer. Since there is no cysteine residue in MBP tag and the protein sequence has only one cysteine, so we can conclude that the dimer being formed is due to disulphide linkage.
Hie Mr. Stanley Ng, the 75 kD band that you have mentioned in your comment is also there on the SDS PAGE gel. I was wondering that may be this is a dimer of cleaved and uncleaved protein, after your comment I am quiet confident that may be I am thinking in a right direction. Thanks !
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression