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
- - - - -

Protein activation after purification


  • Please log in to reply
4 replies to this topic

#1 Makosad

Makosad

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 13 May 2013 - 03:47 AM

I am working on an amidase enzyme, cloned and expressed as a pET conctruct in E. coli, and is origially from a soil bacterial isolate.

I am expressing it as C-terminally fused hisTag and purifying comfortably. Problem is, when freshly purified, the specific activity is around 10 U/mg, and the activity goes upward when I incubate the protein at 30 degrees for approximately one week, upto 40-45 U/mg. After one week, I can use the enzyme in organic solvents for my specific reaction. Additionally, recently i found that Tm of the enzyme also increases form 48 to 55.

Any ideas, what might be happening to protein during that incubation time. There is no truncation in protein happening, no disulfide bond formation indication so far.

kind regards

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 13 May 2013 - 03:54 AM

you may be seeing limited aggregation or disaggregation.

how do you know there is no disulfide bond formation?

tm?
talent does what it can
genius does what it must
i do what i get paid to do

#3 Makosad

Makosad

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 14 May 2013 - 06:27 AM

I did observer some precipitates which I filter/cetrifuge out of the activated fraction. for disulfide bonds, i checked by incubating with DTT and there was no change in activity with time.
Tm = melting temperature of protein

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,616 posts
118
Excellent

Posted 14 May 2013 - 06:51 AM

sometimes you will see an increase in activity of some enzymes when they aggregate a little (might not show up with sds-page, should show in native page).

sometimes you will see an increase in activity when regulatory parts of an enzyme are altered.

the conformation of the enzyme may have changed.

i've never seen tm used with proteins. are you saying that the temperature of maximum activity increased or the denaturing temperature increased?
talent does what it can
genius does what it must
i do what i get paid to do

#5 Makosad

Makosad

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 14 May 2013 - 11:45 AM

we used Tm as an indication of thermostability. so it also become thermostable as the melting temperature of the enzyme increases from 48 to 55. I haven't checked for activity at higher temperatures yet.

Conformation change might be happening, b/c when i performed MS/MS of protein, it was exactly same as the fresh purified protein.




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

©1999-2013 Protocol Online, All rights reserved.