I am trying to purify a bacterial cytosolic protein from E.coli as heterologous host. It has a C-terminal 6xhis-tag. I have previously purified reasonable yields on NiNTA or Cobalt columns, but observed a large fraction (>95%) remains insoluble under regular purification ocnditions in a variety of buffers. I figured these might be inclusion bodies, so tried solubilizing in 8M urea. However, protein still appears in the pellet. Now going to try 6M Gua-HCl, but am wondering about this behaviour. Any suggestions?
precipitation happened after purification? For how long did you contact the solid with urea?
We have not attempted purification so far. We are merely judging solubility of protein after 8M urea treatment of the putative inclusion bodies (IBs). We incubated the IBs for 30 min at room temperature on a roller after resuspension of the pellet.
Hi Wiep,
8M urea, but what else? I assume there must be a buffering component and possibly some NaCl but it would be useful to have more information on the composition & pH of the buffer and what you've tried thus far in case someone else reading has had similar experiences and can contribute suggestions. Also, did you sonicate or use another mechanical disruption method, or purely rocking / rolling of the pellet?
Hi Robin, we use a Tris-EDTA buffer (neutral pH) with NaCl (~200mM) indeed.We tried (without urea) initially also phosphate buffers and Tris-buffers with different pH (pH4-11) - protein was not soluble. Which led us to believe it is in inclusion bodies. We do sonicate (5x30s). To purify inclusion bodies we lyse cells in a buffer that also has Triton and 2M urea, and try to solubilize the pellet in 8M urea (classical inclusion body purification). The last step we do 30min at RT as indicated above.
30 min is not enough for solubilization. Anyway you need to wash the pellet before adding urea to get rid of cell debris.
Thanks Omar, will try longer solubilization, as well as the Gua-HCl (I found in literature it has a broader solubilization activity than urea). We do wash to get rid of cell debris prior to the 8M urea.
Are you sure it is not peptidoglycan? It is not soluble. Treat it with a couple of different kind of lysozymes overnight 4 degree and see if it disappears. I guess the question is really "are you sure it is a protein"? Good luck.
Butch
Bernard, we are sure the pellet contains our protein (antibody, tag), but cannot exclude it also includes PG. Will try to see if preincubation with lysozyme improves the situation.
WKS
Wiep Klaas Smits : May be youcan try adding 1-5 mM betamercaptoethanol or DTT in the buffer containing 8M urea which will help you to solublize your protein
Another option - although it is harsh - would be solubilising with detergents. Sarkosyl and SDS are both possible options if you are not subsequently performing a process that trace detergent amounts would have a bad effect on. If the Inclusion bodies are adequately purified, dissolution in 10% SDS, or 1% Sarkosyl in buffered saline could be an option. The SDS protocol worked well for me with for one very insoluble protein where following complete dissolution of the IB pellet, about a week of dialysis against 500mM NaCl-containing buffer lacking detergent resulted in sufficient reduction of detergent levels that we could bind it to resin and isolate the protein. Detergents can also be depleted using specific ion exchange resins (i.e. Dowex, AG1-X8 or similar) with exclusion sizes small enough to prevent protein binding.
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