Hi. In our laboratory, we produce a recombinant protein in E. coli, but it is generated very contaminated with endotoxins. We want to remove these endotoxins. We have used a Pierce kit called "High Capacity Endotoxin Removal Spin Columns" but it really has not helped us. We have tried buying it twice and even writing to Thermo to check if we have a problem or something and we definitely do not know what is happening because it does not work: the protein before and after passing through the columns has the same amount of endotoxins. My question is, have you ever had any problems with this kit? Do you know what others kits are recommended to solve this issue? which brand? is it better polymyxin instead of polylysine? We have also read that Triton X-114 can be used for the removal of endotoxins before using the columns, but the purification of Triton X-114 seems complicated and that traces remain that can affect the biological tests. Do you know which kits are reliable to completely eliminate the presence of Triton in the protein sample? Thank you.
Dear David
i like a lot endotrap coloumn that where developed by hyglos (http://www.hyglos.de/en/products-services/products/endotoxin-removal/endotrapr-hd.html)
and Now are sold by Lionex, biovenfor or also vwr in us.
you cit on my an find a detailed report about it on my blog: https://proteocool.blogspot.com/?m=1
ProteoCool n°14: EndoTrap: Rapid and efficient EndoToxin removal at PAGE 3
good luck
Manuele
In a previous job long ago (~15 - 20 years), my lab used these kits extensively, but we got rid of them, as we often got rather strange results in immunological tests.
If you have to produce the same or a few proteins all the time and perhaps even in larger quantities, I recommend developing a protein purification process that removes the endotoxin. I assume you work with E. coli, so you are dealing with LPS, which binds quite strongly to anion exchange resins. If you run a gradient at pH 8 from 0 to 1M NaCl, it should elute late in the gradient, after your protein. It's a bit too long ago, but I think LPS comes off at ~500 to 600mM NaCl.
Depending on your protein, you may, however, have the problem that some of the endotoxin binds very strongly. In that case, add a washing step with Triton X-100 or some other detergent after binding your protein to the resin.
Then you have to wash off the detergent. It can stick to the protein like glue. You need patience with this. For some extreme cases, we ran buffer for several hours at low flow rates over the column. I remember in one case we even used 10% ethanol.
Naturally, you need additional column steps to purify your protein, but AEX is usually the capture step.
Dear DAvid
i forgot to tell you that a possible alternative, is also to use the n
https://www.lucigen.com/ClearColi-BL21-DE3-Electrocompetent-Cells/
i tested it several years ago and in terms of protein expression, solubility and growth rate work quite similar to the standard BL21(DE3), however in this case you need to pay more attention from the beginning to use reagents (e.g buffers) endotoxin free during all your process otherwise you risk to re-introduce endotoxin into the sample also if the E.coli strain do not produce it.
Of course in case you are performin purification with Endotrap, you have to be sure that the buffers which are you using for the purification (prepared using endotoxin free water) and the tube for sample collection are endotoxin free.
good luck
MAnuele
Hello Manuele Martinelli, thanks for your answer. Have you actually used those columns? Did they do the job correctly? Thanks a lot for your insights. Yes, we are taking the measurements for that. We checked por endotoxins with LAL all the buffers during the endotoxin removal process with the Thermo columns. That wasn't the problem :/. About the cells, we thought about that posibility, but the recombinant protein is made in another group (in collaboration) so we need to talk about that.
Hello Achim Recktenwald, thanks for your answer. What immunological tests did you use? What problems did you encounter? For example remamants of polymixin or something like that? We are trying to produce one protein [20 mL (2mg/mL) per year aprox]. Yes it is E. coli, and the problem is LPS. Our protein is hydrohilic, veeery stable in PBS at 4°C. We were considering AEX but we don't really have that equipment in our laboratory since we are very preclinical. Indeed the protein production is made in another lab in collaboration, perhaps they can access to that type of procedure. We were trying to use Triton X-114 to remove some endotoxins before using the kits, but it seems like the removal of Triton is also very complicated, and would requiere some other kit.
Thank you both.
Dear David
i used endotrap Hd and endotrap red several times when i was in Novartis Vaccines to prepare proteins for mice immunization and it works very well, expecially the endotrap HD.
good luck
Manuele
Hello David
I found by chance this forum. I am using the same kit "High Capacity Endotoxin Removal Spin Columns" bn these days, and I am experiencing I guess the same problems you descrive. Its efficiency in removing LPS, either from contaminated proteins, or from ultrapure LPS used as control, is not what declared (ranging around 80 %). Sometimes, it does not even work and we do not understand why.
At the end, how did you solve the problem?? Thank you very much, Marco
Actually, do you know other people who experienced troubles with this kit??? Thanks again
Hello, we changed to another brand and it works fine. I can ask the specific name of the new kit if you need it.
Thank you so much, I would appreciate that. We have to remove LPS from small volumes. These columns from Pierce seems working or not working by chance...
thank you
Marco
Dr Cassatella, this is the new kit we are using: https://www.genscript.com/kit/L00338-ToxinEraser_sup_TM_sup_Endotoxin_Removal_Kit.html
ToxinEraser by Genscript.
I hope it works for you as well. This one is kind of like "only one use" (even if they say they are reusable). We tried to use twice the same column and it losses efficacy but we only needed a small amount of sample endotoxin-free so it was better than the previous kit that never worked.
Thanks a lot David., I appreciate it very much. I hope it is sold in italy.
My warm regards from Verona!
Marco
Hi David,
Have you tried EndoTrap columns (or bulk resin)? Specifically the EndoTrap HD is highly efficient at removing endotoxin and getting good recovery rates
https://ilexlife.com/blogs/blog/endotrap-hd-endotoxin-phage-cocktail-uti-treatment
Brad