Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

how do GST-tags affect gel filtration results?


  • Please log in to reply
5 replies to this topic

#1 Kmu

Kmu

    member

  • Active Members
  • Pip
  • 8 posts
1
Neutral

Posted 12 September 2012 - 08:23 AM

I am working with an insoluble protein which is associated with a GST-tag. The tag not only helps in keeping it soluble but also helps in purification. I am however, interested to know if the protein forms higher homo-oligomers. But I was reading about how running a GST-protein though a gel filtration column results in GST-GST interaction. This will not give me the results I wanted to look for. Also, cleaving the tag off makes the protein precipitate out. Please help.

#2 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 12 September 2012 - 12:44 PM

your protein will come out as dimers (GST-GST interaction) after the void volume of the gel filtration. I would do a GST purification step followed by an ion exchange step. Even a second ion exchange. You can do MonoS, dialyze on the other side of the pI and do a MonoQ to get it even purer if the first thing doesn't work. Bottom line: when insoluble protein that is not doable on gel filtration, use ion exchange :)

BTW: as far as I know if a protein is soluble only with GST and precipitates when you cut the GST, then that protein was not soluble in the first place i.e. was not folded in the cell. Are you sure your protein is usable at all? Not that you work to purify it for nothing.

#3 Kmu

Kmu

    member

  • Active Members
  • Pip
  • 8 posts
1
Neutral

Posted 12 September 2012 - 01:24 PM

Thanks a lot !!
I have been facing some issues with the protein's activity. My initial suspect was that the GST-tag was interfering with the assay and so I had tried Factor Xa to cleave it off. I still do not see any activity. Literature shows that it tends to show a very weak nucleosidase activity but the assays done in the paper do not include Factor Xa.
So I am quite clueless right now.
also, I am not entirely sure as to what kind of readout should I expect with ion exchange as I am not really trying to purify the protein further but trying to see if it form higher order oligomers instead.

#4 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 12 September 2012 - 01:33 PM

I am not really trying to purify the protein further but trying to see if it form higher order oligomers instead.


In my opinion, as long as you have GST there you cannot do it.

also if you don't get activity + your protein precipitates after you cleave the tag, be assured that it was born dead. Try different expression conditions aka make an expression test/screen based on activity. It is so nice to work with enzymes that have an activity to check. I would take different expression conditions: temperatures, times, media, inducer concentration and harvest 10 mL culture for each of them, resuspend in 2 mL buffer and sonicate after which spin down to clear the lysate and check some of the lysate for activity. If you don't have activity in the cell lysate, the protein is not folded so don't bother purify it. Just my two cents.

#5 Kmu

Kmu

    member

  • Active Members
  • Pip
  • 8 posts
1
Neutral

Posted 12 September 2012 - 03:44 PM

Thank you for your suggestions. I will try the expression checks. I had done quite a few of them to get overexpression of soluble protein as I took me a while to figure out the temperature to grow it at (overnight at <18C) and the inducer concentration. Plus, addition of protease inhibitor cocktail (for bacterial cell lysate) and the addition of DTT to the cell lysis buffer helped a lot.
However, with all of these I did not check the activity of the protein.

#6 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 12 September 2012 - 09:39 PM

Moderation: I moved topic here from Biochemistry. This subforum is more appropriate for the content of the question.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.