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
- - - - -

No activity of expressed protein obtained from pET32a cloned gene


  • Please log in to reply
6 replies to this topic

#1 hRNA

hRNA

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 01 January 2013 - 03:28 AM

Hi all
I have a 1.6kb gene cloned in pET 32a vector with N-terminal His-tag. For expression I have transformed the clone in Rosetta (DE3) pLysS cells and induced with 1mM IPTG at 0.6 O.D. Then the culture was transfered to 18 C for 10-12hrs. I have purified the expressed protein using His-tag. When the purified protein was loaded on SDS-PAGE, I could see clear expression at expected size (64KDa protein+19KDa Trx tag= 83KDa). But when I perform activity assay I could not see any product. I would like to know, how much essential is it to remove the Trx tag from the protein to get the activity?
It will be really a great help as I am stuck with this since last 2 monthsPosted Image Posted Image

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,417 posts
237
Excellent

Posted 01 January 2013 - 09:17 AM

This is very difficult to predict ahead of time. People often do both N and C terminal fusions in the hope that one or the other will produce active product. Other issues may arise, however. Are you certain the protein has folded correctly? Is your assay working? Do you have a positive control for the assay?

#3 Pangea

Pangea

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 97 posts
7
Neutral

Posted 12 January 2013 - 01:49 PM

Why do you have TRX tag if you are purifying with His Tag? Is it to cleave the His-Tag or what? Are you using ELISA for hte activity assay?

#4 hRNA

hRNA

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 14 January 2013 - 10:59 PM

The protein I am working with goes into insoluble fraction on expression. I need protein in soluble form for its activity assay. The Trx-tag is generally use to get the protein into soluble fraction. As Thioredoxin (Trx) itself is highly soluble protein, it brings any protein in-frame to it in soluble fraction. Later, that tag can be cut from the target protein using enterokinase. thus Trx is not a tag for purification of protein but in enhances the solubility of it. For purification I use His-tag.

#5 HOYAJM

HOYAJM

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 159 posts
12
Good

Posted 16 January 2013 - 11:05 AM

I work with the pET32a vector for all of my expression constructs. I think it is essential that you remove the Trx tag. I believe it is 109AA! A his tag is 6. With the Trx tag you are working with such a different protein I would never predict that it would function like it should in an assay. The beauty of the pET32a vector is you can cleave that tag off.

#6 hRNA

hRNA

    member

  • Active Members
  • Pip
  • 9 posts
1
Neutral

Posted 17 January 2013 - 03:47 AM

Thank you for this help and suggestions.
Ya, you r right that the Trx tag is ~20KDa size. I have cut the tag using enterokinase, but here the problem is, the tag and the enzyme (enterokinase) remains in the digestion reaction alongwith the protein of interest. Get rid of these is problematic for me as it involves further purification which will result in reduction in the amount of target protein (OR may be activity loss!!!!!). Can you suggest me some protocol which does not include lot of downstream proccessing giving maximum yield of target protein without trx-tag.

#7 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 17 January 2013 - 07:24 AM

You can consider to use FPLC systems like AKTA chromatography to purify your recombinant protein after your enterokinase treatment. Use IEX columns.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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

©1999-2013 Protocol Online, All rights reserved.