I am trying to express and purify a human protein in E.coli (His6-SUMO fusion tag). The total protein size is 67kDa with the fusion tag and the protein of interest is 47kDa. The expression is great and about 50% is in the soluble fraction. I usually follow the Ni-NTA purification, size-exclusion, and TEV-protease cleavage. The final buffer is 50mM Tris pH 7.5, 500mM NaCl, 10mM MgCl2, 0.5mM TCEP and 10% glycerol. For some reasons, the final product is not >95% purity. I am seeing clipped products of my protein (~15%) in SDS-PAGE. I use EDTA-free complete protease inhibitors (Roche) during cell lysis and Ni-NTA purification. I have also tried C-term His tag (to check if this is a protein degradation issue) but unfortunately, the expression is very low and I still see the degraded products. Has anyone come across this issue before and what is the best possible way to go about this? Any suggestions would be really helpful.
Have you check codon bias of your insert with the host system. In such cases due to poor representation of tRNAs of that amino acid is underrepresented and may lead to tralsation assembly fall off. So essentially the other bands which you see is not degradation but your protein translated to a certain extent and not fully. Your C terminial tagging results also hints at the same. In such cases host strains which have these tRNAs such as pLysrare. First i would reccomend you to check codon bias in your insert using online softwares then go ahead with these strains.
Hello Venkatraman,
Thanks for your reply. Yes, I ordered the gene codon-optimized for E.coli. Do you still think that i should use pLys rare strains? I have been using BL21 star DE3 cells.
I spent a long time trying to get rid of what I thought was a cleaved version of my protein, and now I am not even sure that the band I was worrying about was really my protein being cleaved. Are you sure this is cleavage product and not just a contaminant?
If you are concerned about metaloproteases (since you are not using EDTA) you can try Roche cOmplete resin (http://www.sigmaaldrich.com/catalog/product/roche/cohisrro?lang=en®ion=US), which is EDTA compatible Ni resin (worked for me to purify protein, but did not get rid of potential cleavage product).
Thank you Alexander. I am sure this is cleaved product as the bands (~4) move down with the protein of interest after TEV protease cleavage.
Okay, are you also confident cleavage is occurring after lysis, instead of during expression? You may want to run a SDS-PAGE or Western of your cells to make sure.
I think it is happening during translation as Venkatraman suggested.
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