Editing Endotoxin Removal

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 NaC'''l. 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 

1. Use endotoxin removal resin at small scale. 

2. Bind to cation exchanger (SP, CM) at about pH 4, wash extensively. 

3. Bind to anion exchanger (Q, DEAE) at about pH 8.0. Elute protein 

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) 

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=== Sartbind Q &amp; S<br> ===

Using the '''strong basic '''ion exchanger type '''Q''' and a buffer '''pH lower than the pl '''of the protein, '''endotoxin will be bound and protein will''' '''pass through''' the membrane (negative chromatography). Using the '''strongly acidic''' ion exchanger type '''S''' and a buffer '''pH lower than the pl '''of the protein, the '''endotoxin will ''''''pass through''' and the '''protein will bind''' and can be eluted in the next step.<br>

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=== Q, Q-S in series&nbsp; strategy:<br> ===

The first Q membrane is used as a product capture step, and the second Q is a negative chromatography step. The pH condition should be optimised<br>in order to let the product run through the second Q and directly captured on a cationic exchanger Sartobind S membrane (S)<br>

'''Example'''<br>

1. Adsorption and elution from '''Q'''<br>– Bind at pH 8.0<br> – Wash with 50 mM NaCl at pH 6.5<br> – Elute with 100 mM NaCl at pH 4.7<br><br>

2.&nbsp; Flow through '''Q''' and loading of '''S'''<br>– Load onto Q and S in series at pH 4.7<br>– The protein flows through Q and binds to S<br>– Disconnect Q from S membrane<br>

3.&nbsp; Elution from '''S'''<br>– Elute at pH 5.5<br><br>

4. Cleaning<br>

1 N NaOH and 1 N HCl on the anion exchangers (Q) and 1 N NaOH on the cation exchanger (S).<br>

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pH: 0.5 to 1.0 pH unit below the PI (isoelectric point) of the target protein (flowthrough)<br>Buffer with low conductivity: up to 200 mM NaCl.<br>Endotoxin removal can be achieved at 50,000 to 30,000 EU/ml: up to 1.5 mg endotoxin (15,000,000 EU) per ml Sartobind Q membrane Device recommendation at start of experiments: 

<br>Sartobind Q SingleSep nano 1 ml<br>Flow rate: 30 membrane volumes/min or higher<br><br><br><br><br>

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[[Category:Protocols]]