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
0
High Yield Protein Expression 10g/L
Started by Pangea, Jan 03 2013 10:06 AM
Expression Protein Yield
5 replies to this topic
#2
Adrian K
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
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
..."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
-
- Active Members
-
- 95 posts
Enthusiast
6
Neutral
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
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
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
..."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
-
- Active Members
-
- 95 posts
Enthusiast
6
Neutral
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
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
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.
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
..."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
Also tagged with one or more of these keywords: Expression, Protein, Yield
 |
Protocols and Techniques Forums →
Molecular Cloning →
Promoter distance from ATGStarted by Guest_miST32_* , 17 Jun 2013  Promoter, expression, construct
|
|
|
|
|
|
Protocols and Techniques Forums →
SDS-PAGE and Western Blotting →
5x SDS lysis bufferStarted by Guest_JoeSus_* , 31 May 2013 lysis, buffer, protein
|
|
|
|
|
|
Protocols and Techniques Forums →
SDS-PAGE and Western Blotting →
SDS Page Problem: Proteins stuck at stacking lineStarted by Guest_Randy Smith_* , 30 May 2013 sds page, western blot, protein and 2 more...
|
|
|
|
Protocols and Techniques Forums →
Biochemistry →
protein fractionationStarted by Guest_salmonela_* , 09 May 2013 protein, fractionation, bacteria and 2 more...
|
|
|
||
Protocols and Techniques Forums →
General Lab Techniques →
break disulfide bonds to study fluorescenceStarted by Guest_Mulligan_* , 27 Apr 2013 Biophysics Fluorescence and 4 more...
|
|
|
