Hey guys,
I've recently cloned a protein into a vector and attempted expression. Colony PCR and gels confirmed an insert of an appropriate size was in the colony that I selected for protein expression. I expressed in C41 cells and ran a SDS gel of the sonicated cells, both supernatant and pellet. There was a small band corresponding to where my protein was meant to be, 60kDa but it was very faint. A much stronger band appeared at ~10kDa.
I ran a his-tag purification and eluted by step elution. About 18mgs of protein eluted, too high for non-specific binding I thought. I ran a gel and got a similar result. A strong band at ~10 kDa and a series of weak bands around the theoretical protein mass.
So is it possible that the protein is migrating a much lower MW on a gel? I thought it may be proteolysis but I added protease inhibiters prior to sonication.
Something with a HIs-tag is binding to the column in abundance. However a gel shows a huge band at 50 kDa lower than it should been. I will of course run sequencing on the plasmid to make sure I have what I think I have but has anyone else got any suggestions as to what may be going wrong?
Any help would be much appreciated.
Best regards
Harry
Just to clarify. Are you saying that you expected to get a 60 kDa band but are getting instead 50 and 10 kDa bands, or are you getting only the 10 kDa band?
The best way to troubleshoot this problem would be offcourse first compare the sequencing results. If you have an antibody available against the protein of interest then do a western blot. Probe the samples with anti-his antibody the oo. Depending on the western results, you may be able to figure out where is your protein runs on a gel, whether the 10kda is a truncated product or it is just a side product which for some reason expresses well along with your protein of interest.
Sorry to ask, but are you sure that the 10 kDa band is not just every small protein impurity running together at the dye front?
Dear Harry
I suggest you to study your protein: check the aa sequence, the isoelectric point , how many cys-cys , if your protein is rich in basic or acid amino acids, what is the function. If the isoelectric point of your protein is close to the SDS running buffer, it can precipitate in the gel showing an abnormal migration. Basic and acid proteins show a fast mobility in Laemmli SDS-PAGE system. Cys-cys rich proteins migrate faster because coiled, add fresh b-Me when you load the samples in the gel. If your protein binds to something, it can even be its binder. Your protein can be cut by proteases, try to express it in different E.coli strains. Run a western blot with an anti-his Mab to identify your protein and to easily eliminate the short molecular mass protein use a dialysis cassettes with 20 kDa cut-off ( Pierce). Good luck
Hi Harry,
You didn't say if it was a fusion protein and the source of the gene.
If it is an N-terminal fusion protein make sure that your gene is cloned in-frame (that error should be obvious with your sequence data) as this could give you expression of the his-tagged fusion only.
If your protein is from a eukaryotic source you may have a run of low codon usage codons that will prematurely truncate your translate. Try it out with Rosetta or similar cells and see if they make a huge difference. If your expecting toxicity (hence the C41 strain) you can always prep the Cm-resistant pRARE from Rosettas and co-transform into C41.
What plasmid are you using? If it's T7-based you have to use cells containing the DE3 lysogen.
Do you have a good protease inhibitor cocktail in your lysate?
Have you checked the insoluble fraction for your full-length protein?
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