A problem in Large scale protein expression

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syawash
syawash's picture
A problem in Large scale protein expression

Hi,
I am using BL21 and pETM-41,pGEX to express my protein. I have got the desired band on the SDS page when I do small scale but I can't find my protein in large scale.
I would be glad if somebody help me.
Thank you very much.

yang
yang's picture
syawash wrote:Hi,

syawash wrote:

Hi,
I am using BL21 and pETM-41,pGEX to express my protein. I have got the desired band on the SDS page when I do small scale but I can't find my protein in large scale.
I would be glad if somebody help me.
Thank you very much.

maybe your protein degraded when you scale.

syawash
syawash's picture
Dear Yang

Dear Yang
Thank you for the reply. How can I do to solve the problem?
Thank you very much.

Best,

R Bishop
R Bishop's picture
How many times have you

How many times have you repeated the scale up?
The only possibilities are
1) you didnt do everything exactly as before in the small scale

2) degradation. - could be cured by adding protease inhibitors most likely.

Im surprised you would get degradation in large scale and not small. To me this suggest your conditions for expression were not quite right. Can you post your small scale and large scale protocols so that we can compare them? That might help others dig into your problem. Finally were your bacteria growing at the same phase as your small scale pilot experiment?

Good luck

Rb

Add colour 2 ur life
Add colour 2 ur life's picture
as u said in small scale u r

as u said in small scale u r protein was expressing, r u following same initial od and induction od?? iptg concentration all are same in large scale??
start with initial od and induction od 0.6-0.8
your protein is soluble or ib??
r u checking the pellet as well as sup??
just make sure that all u r doing right and put other details here then we can do something..

all the best