I want to produce a recombinant protein about 65 kDa by BL21 E.coli and purify by Ni-sepharose beads (GE healthcare). (I fused myc and his tag at C-terminal end of my protein)
After elute my sample with elute buffer contains 250 mM imidazole, I dialysis my protein to PBS (pH 7), enhance my protein concentration by 50 kDa condensor, run 10% SDS-PAGE to confirm the purity of recombinant protein and storage at -20℃.
I detected a major band between 55-70 kDa in molecular weight ladder (I think that is my protein)
But I cannot detect the protein signal by Western blot no matter using anti-myc or anti-his tag antibody.
I think there is something wrong in my recombinant protein, but I don't know what's happening.
I have checked the plasmid contruct by restriction enzyme digestion, primary and secondary antibody's function by positive control.
Is there any possible protein degrade only at C-terminal of protein?
One possibility I can think of, assuming that everything is ok with your western blot, is that your tag is getting cleaved off in the link connecting the C-terminal tag with your protein. Most times there is a cleavage site to give the option of cleaving after purification. If that's the case, you could increase the concentration of protease inhibitor. Alternatively, although longer, you may mutate the link to get rid of that site.
Of course, this is just one possibility.
First of all, you have to be sure that the band you observed at 55-70 kDa is really your protein. USe bacteria transformed with an empty vector to prepare a negative control that you can load on the SDS-PAGE close to your sample.
I am agree with Paul Lesbat that the use of a 50 kDa condensor should not be the best if your regular protein is about 65 kDa. Try to use filter with a cut-off about half of your protein to be sure to keep it on the filter (e.g. 30 kDa units). Another drawback should be that if you concentrate a lot, your protein might adsorbed on the filter.
As a good control, ou can try to load a sample at each step of your procedure on the same SDS-PAGE i.e. from left lane to right lane MWM / crude extract of your negative control / crude extract of your expressing bacteria / Flow-trough fraction from the Ni beads / eluted fraction / dialysed sample / concentrated fraction / filtrate from the condensor. Do a Coomassie Blue and a western-blot. It will help you to know where is your protein.
To answer your first question, it is right that protein can degrade only at the C-terminal. There could be a particular "sensitive" sequence on your protein C-terminal that should be a nice target for ndogenous proteases (a large loop, a flexible region, particular amino-acids patches...).
In my experience you may have a cleavage of a C-terminal tag and this is most likely what is happening in your case. Can you tag your protein at the N-terminus? If you can't for any reason, you may also consider internal tagging. Choosing a site for internal tagging can be tricky, but it could be easier if you have some information about the 3D structure or at least the secondary structure of your protein.
Proteases are found everywhere. Serine proteases are frequently available in cell which can cleave at any place with in the protein, leaving small fraqments. You can read my article entitled "Proteolytic inventory of Thermococcus Kodakarensis".
It may possible that some endopeptidase is cleaving your protein. It may possible that any C-terminal peptidase degrade protein on C-terminal, but there is less chance for that. I suggest try to use 2-5 mM of EDTA in your buffer throughout the process in order to avoid these proteolytic activities.
Thank you Paul for correction. For Ni-sapharose, EDTA can not used as protease inhibitor. but PMSF inhibits the serine proteases notC-terminal peptidases (Carboxylicpeptidase). You may use PMSF along with the inhibitor for carboxylicpeptidase.
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