When producing recombinant proteins in E. coli and other gram negative bacteria it is often necessary to remove bacterial lipopolysaccharides (LPS) from the final product. This is especially important when doing immunological readouts and when developing manufacturing processes as endotoxin can cause false readings in cell based assays and there are limits to the amounts of endotoxin allowed in human products.
LPS is hydrophobic due to lipid tails and has a net negative charge at slightly acidic, neutral and basic pHs. These properties can be utilized when trying to remove endotoxin from protein. If the protein starts out as an unfolded molecule like those formed in inclusion bodies one can redissolve the inclusion bodies in an appropriate chaotropic buffer such as 8 M urea and bind the protein to an ion exchanger. At pH=8.0 on a Q-based matrix most proteins will elute once the salt concentration has reached 300 mM NaCl. Endotoxin on the other hand does not come off the matrix until about 0.5 M NaCl. Even more efficient, if your protein binds, is the use of a cation exchanger at pHs of around 4. LPS is still charged and does not interact with the matrix, while a lot of proteins will bind. The advantage of this binding is that the matrix can be stringently washed with either buffer alone or a buffer with added detergent. The above procedures will also work in the absence of chaotropic agents, but not as well as the interaction with the protein sometimes is too strong to remove with just buffer washes. The use of a cation exchanger with detergent usually solves this problem. Another method for the removal of endotoxins is the use of hydrophobic interaction chromatography. If an efficient purification scheme using HIC can be worked out in which the protein of interest elutes from the matrix the endotoxin is usually left behind tightly bound to the column. Reversed phase chromatography is not a common method for getting rid of endotoxins as the matrices quickly get loaded with the lipids and then bleed endotoxin for a long time unless very diligently washed.
There are currently resins on the market specifically for use in removing LPSs. We have not had very good luck with any of these as usually either the protein binds as efficiently to the matrix as the endotoxin and cannot be recovered easily, or both endotoxin and protein elute in the flow-through fractions.
For very difficult proteins reducing the protein prior to application to the column in combination with the use of a non-ionic detergent in an 8M urea buffer seems to work quite well. An excellent alternative is also phase separation with Triton X-114. To a concentrated protein solution in a non-chaotropic buffer add about 1/20 th volume of Triton X-114. Cool and agitate the sample to get dispersal of the detergent into the liquid. Heat to about 42 degrees C and allow the phases to separate by centrifugation. The protein solution can then be aspirated from the detergent phase and is often devoid of endotoxin. If not the procedure can be repeated indefinitely. The disadvantage here is that residual Triton will reside in your sample and can not be dialyzed out (micelles) - you will have to perform some sort of chromatography to get rid of it. Reversed phase works quite well for this.
An algorithm of things to try is:
1. Use endotoxin removal resin at small scale. Works? Great. If not:
2. Bind to cation exchanger (S matrix etc...) at about pH = 4, wash extensively if endotoxin is still present after elution, go to:
3. Bind to anion exchanger (Q matrix, DEAE...) at about pH=8.0, elute protein, test for endotoxin. Still stuck?
4. Reduce protein with e.g. DTT, dissolve in an 8M urea buffer, bind to cation exchanger. Wash extensively with 8M urea buffer containing 0.5% Triton X-100. Exchange into 8M urea buffer without the detergent, elute as usual (on-column refolding can also be tried at this point)
5. Still there? (This one is tough then) Try repeated extractions with Triton X-114 until no more endotoxin is present or develop a method using HIC.
6. Still having problems? Write to the Proteinchemist.