I'm working with an integral membrane protein that we want to express and purify for structure/function studies. The full-length gene is cloned into a pET vector with a C-terminal His6 tag. We get fantastic expression in E. coli C43(DE3), but when blotting lysates and crude membrane fractions, there is only weak blotting at the top of the gel/membrane.
When membranes are re-solubilized in detergent and assessed by SDS-PAGE, there is a large and prominent band just smaller than the expected size of the recombinant that does not bind a Ni column. Several prominent, high MW bands are present in crude membranes and to a lesser extent in detergent-reconstituted membranes. Digest and LC/MS of the soluble species confirms that it's the recombinant protein, but C-terminally truncated so lacking the tag. The truncated region contains an important active site motif, so the truncated form is also inactive in assays when purified.
I have a few ideas to obtain the full-length form (cell-free translation, denaturing purification) but was curious if anyone had experienced something similar and had used an approach to prevent truncation in a similar case. We can map the likely site of proteolysis from the LC/MS data, but not sure what to do with that information.
Two things spring to mind. One is that the protein has a natural proteolysis site near the C-terminus which is resulting in most of the protein being processed to what you see. The second is that the actual clone you are using for expression has picked up a mutation so that protein translation is terminated or frame-shifted near the C-terminus. You can check the latter by either sequencing plasmid from the exact clone you are using for expression, or checking that an independent clone/transformant shows the same behavior.
Hi Michael J. Benedik and thanks for the reply.
We of course Sanger sequenced the plasmid after cloning and it looked just fine, but good to check it again. This happened on multiple clones but we'll have the full plasmids nanopore sequenced to make sure we didn't pick up a problem earlier on that propagated. It seems unlikely to me that proteolysis would result in a singular, well-resolved species.
Have you looked for motifs recognised by proteases? If you find them, you can design a mutation to remove the cleavage.
Hi Piero Pileri
The protein is not in the MEROPS database. I have a pretty good idea of the site of cleavage based on the fragment size and peptide LC-MS data though. Can you suggest a tool that might identify specifically the scissile bond and/or the likely E. coli protease that can cut it? Otherwise, I am stabbing in the dark when replacing residues.
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