I am currently trying to purify a protein called MMP-2. It has an approx. molecular weight of 72 kDa. For the past several weeks, I have been purifying this protein expressed from BL21-RIL E.coli strain cells. I harvest the cells, lyse them using sonication, then isolate the insoluble pellet. I then use 8M urea buffer to dissolve the insoluble pellet and pour the mixture (after incubation with Ni-NTA resin for 2 hours) into a glass column.
I refold the protein on the column using a matrix-redox protocol (containing buffers with 20 mM imidazole) and elute the protein using 500 mM imidazole at the end. Once this is complete, I dialyze the solution against low salt buffers w/ DTT and EDTA to inactivate the protein (since MMP-2 is a zinc-dependent enzyme). Then, I run the dialyzed mixture through a 50 kDa GE Healthcare Vivapsin 20 filter and run my sample on a SDS-PAGE gel.
Once done, I stain with coomassie but I see 2 bands showing around 20 and 25 kDa. I did mass spec and the guys said it was MMP-2?? But under the bacteria E.coli domain, it came under FKBP. I tried adding more EDTA (1 mM -> 5 mM) but still getting these bands. I also tried filtering the solution through the 50 kDa filter again but still showing on my gels!. So, is my protein creating a complex with FKBP?? Please help.
The obvious answer seems to be that the 20 and 25 kDa bands are proteolytic fragments of MMP-2. I don't understand why you think they are FKBP.
I don't understand your comment about the mass spec. Could it just be the pro domain or other processed product of MMP2. And is your protein even active. Normally people don't prepare MMP2 this way. Sometimes when proteins are expressed in E. coli, re folding proteins like HSPs and FKBP co express. But it is likely it is just a Mmp2 fragment
Not necessarily. If Mmp2 is a diner, or mulitmeric, the fragments could co purify because the pieces are binding to the intact protein. I think Mmp2 is mulitmeric.
Yes but MMP-2 can be expressed as a dimer, but around 140 kDa, which doesn't make any sense since I haven't seen anything that high on my gels. If my fragments are 20 and 25 kDa, it doesn't add up with my main 72 kDa band. I heard FKBP proteins can bind to zinc metal ions as well. Given that MMP-2 has a Zn2+ catalyctic domain, wouldn't it bind to that during the refolding process??
Several points:
The efficiency of filtration of a 25 kDa protein with a 50 kDa MWCO filter is not very high, so you could not count on complete removal, even if the 25 kDa protein is monomeric.
The molecular weights of the proteolytic fragments you see on a gel do not have to add up to the molecular weight of the intact protein, because small peptides that are cleaved off may run off the gel and not be observed.
On SDS-PAGE, non-covalent complexes are separated by the denaturing action of SDS. If you started with a non-covalent dimer of 72 kDa MMP-2 monomers, you would not expect to see the dimer on SDS-PAGE, only the monomer.
Reiterating Marcia, a protein can undergo limited proteolysis without falling apart. This you could have an apparently intact 72 kDa protein with a few sites of proteolysis, but when you run it on a gel it is separated into a set of smaller pieces.
The Zn2+ in the catalytic domain of MMP-2 would not be likely to bind to another Zn2+-binding protein, even during refolding, because the Zn2+ ion would be coordinated to amino acids and occluded. It is more likely that the 2 proteins would compete for the supply of Zn2+ ions, if there were a limited supply. Moreover, unfolded proteins won't bind Zn2+ until they have refolded to some extent such that the amino acid residues responsible for Zn2+ binding are correctly positioned.
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