We highly recommend the EndoTrap® endotoxin removal system because it is the most reliable method to remove LPS from recombinant proteins after using an E.coli expression system.
We use the Profos EndoTrap® Red columns for the removal of LPS originating from E.coli (BL21DE3) expression of recombinant proteins. We are only interested in removing LPS from E.coli from our samples, but the system also removes LPS from Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens and Citrobacter freundii. We had some difficulties with protein recovery from the column at first because we used the EndoTrap blue columns which are not suited for PBS-buffered samples. When we contacted Profos customer service, they were very helpful and suggested that we use the red columns instead. In addition, they provided a free sample of this column. We found that protein recovery was very good (~90%) with this type of column. We also tried different removal methods with far lower protein recoveries.
Since we needed a reliable LPS-free protein preparation for in vitro T cell proliferation assays, we ran several tests with the EndoTrap® Red columns to be sure that the LPS was efficiently removed. The LPS-sensitive cell line chicken HD11 did not produce NO– after incubation with the LPS-free protein; this result indicatates complete removal of LPS as there is no LPS left to induce cell stimulation or proliferation. In the “LPS gold standard test”, the Limulus amebocyte lysate test, LPS levels were below 0.8 endotoxin units/ml in the most concentrated sample. In a silver stained SDS gel of our purified protein, no degradation products of our protein could be detected. The protein worked fine in the bioassays and for immunization after the LPS removal procedure. We also used the purified protein in ELISAs and Western blots.
The procedure itself is very fast and convenient. The columns are prepacked and the kit also provides all buffers needed. After washing the column, an equilibration step follows and then comes the elution of LPS free protein. The column can be reused several times with same good results. The preparation of a 10 ml sample takes about 5 hours.
Profos customer service is very friendly and helpful and solved our initial, self-made problems (when we chose the wrong columns for our application). We compared the EndoTrap® Red columns with several other LPS removal procedures and found this to be the best system in our hands.