I'm purifying a protein that is fused onto an MBP tag (42.5 kDa). My protein size is roughly the similar size of the tag (47kDa). Together, they form a complex about 89 kDa. After a run through a dextrose column, I've visualised my proteins on an SDS gel and obtained a band corresponding to rthe right size.
Next is the cleavage of the tag from my proteins. I've optimised the conditions of optimization to 1% of Factor Xa+ 0.005% SDS to the protein concentration and a reaction of 6 hours at room temperature. I've checked the sequence of my protein and there are no sites where the enzyme will cleave my protein internally. After cleaving the fusion of my proteins with Factor Xa, I run the sample through the dextrose column again to acquire a sample that contained only the Factor Xa and my protein.
However, when I visualised my proteins on a gel, I got bands that correspond to the fusion protein. I have no idea why this happens and I don't know if there isa possibility of my protein self associating into a dimer form without the tag.I do have very faint bands that correspond to the size of my proteins but this is fainter than the bands that are twice the size of my proteins. The size of the enzyme is 45 kDa.
Anyone with ideas with what is happening?
thanks
Hi there,
First issue to consider: Is the protease actually active? If yes, is the amount used enough for full digest? So condition can be adjusted (amount and time of incubation). And finally is my fusion actually substrate? If cleavage site is hidden, no cleavage occurs. So adding low concentration (ie non denaturing concentrations) of detergent or chaotropic agent might help to uncover the cleavage site.
Trying to interpret the gel: Were there lane 5-10 descriptions? Was the gel analysis run reduced versus non-reduced? Is the main main band (~80 kDa) a doublet? Lanes 9 & 10 don't seem to have the 38 kDa lighter intensity-band but have the lighter intensity-bands seen between 38 and 80 kDa.
Do you have a positive control for your cleavage? The fusion is in the soluble fraction versus solubilization of product in inclusion bodies, right? After the first affinity step, before cleavage, can the fusion molecule be tested for activity? Are there cysteines in this protein? There may be no internal factor Xa sites in the construct but could there be endo/exo protease liabilities from the host? In other words, is your protein intact conformationally and in contiguous length?
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