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
- - - - -

High Yield Protein Expression 10g/L

Expression Protein Yield

  • Please log in to reply
5 replies to this topic

#1 Pangea

Pangea

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 97 posts
7
Neutral

Posted 03 January 2013 - 10:06 AM

Hi,

how can İ best get in E. coli or Mammalian cells an protein expression yield of more than 1g/l?

Any kind modifications?

Cheers

#2 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 05 January 2013 - 06:32 AM

How do you optimize your expression in the first place?
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

#3 Pangea

Pangea

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 97 posts
7
Neutral

Posted 05 January 2013 - 06:47 AM

It will be a pET vectorsystem for E coli. One can play with media and ZnCl2, Glucose and VPA. But is that enought for very high yield production.?

#4 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 13 January 2013 - 08:11 AM

I tried media replacement before induction. It is relatively yields higher expressed product than just merely induction with old media with / without modification.
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

#5 Pangea

Pangea

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 97 posts
7
Neutral

Posted 13 January 2013 - 09:30 AM

So you mean replacement by centrifuging the cells and renewing the media. Can you give me more details like an protocol?

#6 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 13 January 2013 - 02:04 PM

Yes.

Based on my memory, First I grow my E. coli till it reaches a certain OD600 value I want (I usually grow it to 0.5, some protein I still can express it very well at 1.0), centrifuge 5 minutes at 4'C with 8000g to pellet the bacteria cells,
I will then replace the media with final concentration of 0.5mM IPTG to induce the protein expression. My cells growing usually took 3-4 hours (37C), and protein expression usually took another 4 hours (~30C).

p/s: When I am Growing the cells I do include antibiotic. But when I do expression, I excluded it totally. Haven't really verified by myself, but I believe production of lactamases will reduces the availability of amino acids to be use in making the recombinant protein.
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.