Replies

• 1

  John LaCava

  19 November 2008 | 05:08

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  Hi,

  We’ll need more info.

  Let me know the following:
  1) Which organism you want to purify from?
  2) protein is cytosolic or what?

  Cheers,
  John

• 2

  Ana Melo

  19 November 2008 | 10:10

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  Hi John,

  The organism is E. coli and the protein is membraneous.

  Thanks and cheers,

  Ana

• 3

  John LaCava

  19 November 2008 | 15:32

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  So… the membrane proteins can be quite tricky I think. Although, I dont have great experience with them.

  As I understand it, solubility is an issue.

  Can you say how soluble is your protein once released from the membrane? Normally you will need to use a lot of detergent in the extraction buffer to keep the protein happy and soluble (hydrophobic patches covered by detergent I suppose). The usual suspects would be Triton x-100, deoxycholate & sarkosyl.

  These conditions may not be compatible with your favorite affinity-tag (for purification) depending on the concentrations. You’ll need to check what information is available on this. I would start by having a look at Protein-A-tag and HIS-tag, these are common and often with reasonably stable binding even in stringent circumstances.

  Biotin / Avidin could be a choice, but elution from the column becomes an issue… but may not be needed as you could try your enzymatic assay on the column.

  There are many many tags out there, so you’ll have to find good extraction conditions for e. coli membrane proteins first, then a tag best suited for those conditions.

  Another approach is to over-express it A LOT so that it goes (on purpose) to inclusion bodies. Those are easily separated from soluble cell material… then you dissolve that in urea and slowly re-nature the sample… could make purification more simple… and will have a high yield… although the specific activity of the prep may vary (and may not be excellent, hard to say without trying it).

  As for the lysis method itself… common ones for e. coli are sonication (I like this method, it is very effective when done right) and french press (I have never used one, but they are popular). There are also some reagents out there like the BD Bug Buster reagent. I played with this for yeast, and it worked well enough… it certainly is simple and fast.

  Hope this helps get you on your way.
  I can send you some refs on high efficiency sonication lysis if you decide to go that route.

  Best wishes,
  John

• 4

  Ana Melo

  19 November 2008 | 18:13

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  Thank you very much once again.

  I’ll have to think about it very carefully, my goal is to prove interaction between to proteins, so I have to use mild treatments.

  Best wishes