purification of non his-tagged proteins - (Aug/01/2008 )

Hi all,

i need help and advise bcs am new in expressions, have any body idea about how can purify non his-tag proteins *without)
bcs i have construct without his tag

thank u in advance

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If at all possible I think it would be easier to put a His tag on your construct, it will allow an easier method of purification (especially if you are new to this) and it makes checking the purification easier as you can western very easily with a His antibody.

If you have a good antibody to your protein you could try making an immuno-affinity column, but probably very expensive.

I think the initial effort of putting a tag in your construct will save you loads of pain and effort later.

All the best

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Some proteins like synuclein can be purified thru FPLC using different columns. Its tedious but one does get a lot of protein.

Easier way would be to add the his tag or any other tag and purify it.

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you could try ion exchange or size exclusion, or even one after the other. It isnt as specific as a affinity purification, but it works.

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Hi

You have several options: ammonium sulphate precipitation, ion excahnge or hydrophobic interaction chromatography. There are probably other options too but they will be specific to particular protein types. These methods are genrally applicable to all proteins assuming the protein is soluble enough in the buffers required.
You will probably need several purification steps and should therefore test/optimise each one with small scale tests before trying a large prep.


Ammonium sulphate preciptitation

This is an easy way to clean up a lot of your protein before you move on to using purification columns. What you want to do is do small scale tests where you add increasing amounts of ammonium sulphate, mix at 4oC for 30 min and then centrifuge to collect the precipitate. Run the supernatent and precipitate on a gel (you will need to dilute the sample with water or lysis buffer to reduce the ammonium sulphate so that the gel runs ok).
What you want to find is the amount of ammonium sulphate which is just too low that your protein of interest stays in solution. Then in future you can add this concentration of ammonium sulphate, precipitate contaminating proteins that are less soluble than your protein of interest and centrifuge to remove them. To the supernatent you can then add more ammonium sulphate that is just enough to precipitate your protein but non sufficient to precipitate all of the remaining contaminants. This time you keep the precipitated protein pellet and discard the supernatent. The protein can then be resuspended in a buffer of your choice and dialysed agaisnt more of this buffer to remove the ammonium sulphate.
You are unlikely to remove all contaminants using this method but it will quickly get rid of a lot of contaminating protein.


ION EXCHANGE

Don't use a high concentration of protein for this or you will precipitate everything in low salt buffers that you need in order to stick the protein to the column. Keep the protein solution below 1 mg mL-1 if not below 0.1 mg mL-1.

You can buy HiTrap IEX Selection Kit from GE life sciences which has 7 different ion exchange resin columns. You should look up the calculated pI of your protein based on the protein sequence (use Expasy tool "ProtParam" http://ca.expasy.org/tools/protparam.html)
Then if your protein pI < 7 try Q-sepharose first or SP sepharose if pI > 7.

You should try different pH conditions (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9) for the buffers to find the pH where the protein will just bind to the column. i.e. for Q-sepharose (anion exchange) find the highest pH where the protein of interest binds where as for SP sepharose (cation exchange) look for the lowest pH where the protein will bind. Then use a linear gradient from 0 to 1M NaCl in your buffer of choice (usually something like 20 mM Tris or 20 mM sodium phosphate) to elute the protein. Set this linear gradient to run over 20 column volumes. I.e. for a 1 ml column = 20 mL, 5 mL column = 100 mL.
This is discribed in detail on pages 30-36 of the GE life sciences Ion exchange handbook which can be downloaded from their website.
http://www4.gelifesciences.com/aptrix/upp0...moduleid=166190
I suggest you read the whole manual though if you are new to this technique.

Run fractions on a gel to find the fractions that contain your protein of interest.


Hydrophobic Interaction Chromatography (HIC)
Again GE life sciences sell an HIC selection kit (Cat No 28-4110-07). Read the instruction manual. These columns bind protein based on a hydrophobic interaction instead of the charged interaction used by ion exchange. Test different resins on a small scale to find the only that gives the best separation between the protein of interest and contaminants.


These columns generally give worse separation than ion exchange so it is normally better to use these columns after doing an ion exchange step. You normally want to get your protein from cell lysate to pure as fast as possible to prevent contaminating proteases from chopping it up.


GENERAL CONSIDERATIONS

Make sure you remember to keep everything sterile to avoid introducing a contaminating protease from your own skin, non-sterile water...
I.e. sterile filter all buffers into autoclaved bottles, wear gloves - especially when touching fraction tubes and sterile filter your protein solution once you finish the purification. Work at 4oC wherever possible to reduce proteolysis reaction rates.

Even if your protein looks completely clean on a coomassie stained gel when you purify it, I suggest keeping a small amount of the protein solution on your lab bench (at room temp) and taking a sample for SDS PAGE every few days. Then after a couple of weeks run these samples on a gel and see if your protein really is stable or not. If it is being degraded you still have some protease contamination and you need to add a further purification step.

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