I need to use my recombinant protein in cell culture with T cells but it has been produced in E. coli and has LPS.
Rosa,
To remove LPS there are lots of procedures out there, but I have found the best is to use a Q column (anion-exchange) with your buffer system chosen so that the protein of interest is below its isoelectric point, if possible. The positively-charged protein should not bind to the column, but endotoxin (as well as any residual nucleic acids) should bind. One advantage of this approach is that you shouldn't dilute your protein by much as it will pass straight through the column, and you can use Hi-Trap or similar small columns which can be used with a simple syringe. Some manufacturers also make specific filters (Mustang is one type) and endotoxin-removal resins, which I have used in the past with variable results. To measure endotoxin, use a LAL assay - there are lots of these commercially available as kits. Hope this helps, Dave
Rosa,
To remove LPS there are lots of procedures out there, but I have found the best is to use a Q column (anion-exchange) with your buffer system chosen so that the protein of interest is below its isoelectric point, if possible. The positively-charged protein should not bind to the column, but endotoxin (as well as any residual nucleic acids) should bind. One advantage of this approach is that you shouldn't dilute your protein by much as it will pass straight through the column, and you can use Hi-Trap or similar small columns which can be used with a simple syringe. Some manufacturers also make specific filters (Mustang is one type) and endotoxin-removal resins, which I have used in the past with variable results. To measure endotoxin, use a LAL assay - there are lots of these commercially available as kits. Hope this helps, Dave
Thank you David.
That aproach could solve the problem of specific binding between my protein and LPS
Columns based upon polymyxin B resin are often used to remove endotoxin (Pierce, Sigma), Lipid removal Absorbant (LRA Sigma?) is supposed to be used commercially
Triton X-114 phase separation is used as is activated carbon/charcoal.
There is now a modified E.Coli that does not produce a bioactive LPS (ClearColi Lucigen)
http://www.nature.com/nmeth/journal/v10/n9/pdf/nmeth.f.367.pdf
Ian Teo
I have just used polymyxin B resin to shake with my protein solution in a cold room (I have tried different times and just checked LPS content in a protein solution - for my solution the best time was 2 hours of lazy shaking), then I have just centrifuged resin - it was easier to do than with a column but it worked well without diluting my protein solution. And I have used Limulus assay (LAL) to check the level of LPS before and after cleaning step but it was a pain to use. I have seen advert of EndoLISA Endotoxin Detection Kit, and I think I would try it before going once again to Limulus assay. Or maybe LAL assays got easier to use in the last 10 years?
Hi! I am also trying to eliminate endotoxin from a protein sample. My problem is that my protein is so big that it gets trapped in the columns (it is a bacterial fimbria). Does anyone know about a column that works for high molecular weight proteins or some kind of beads that I can use in well, so that I don't have to centrifugate the protein in a column? Thanks!
Hi Alejandra,
Maybe you can find the Q-resin in bulk format and perform the anion exchange in solution with steps of centrifugation instead of column....
I suggested buying endotrap blue before from Hyglos. All you have to do is pass your protein through a 1ml column. Then you are done.
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