insoluble protein expression help - (May/05/2011 )
Hi everyone. I hope you guys can help me troubleshoot my problem.
I cloned my insert into pMAL c5x expression system and transformed it into NEB express E. coli. About a year ago I was able to express the protein in the soluble fraction. But recently I needed more protein so I started protein purification again. However, not only is my protein insoluble but almost all the protein is insoluble. I followed the same procedure I did a year ago ( 13 hour overnight culture; Induce at OD = 0.5; induce for 4 hours with 4mM IPTG). So far some trouble shooting tactics include. Making fresh AMP stock,IPTG stock, induce at lower temperature, lower IPTG concentration, and using a lysis buffer instead of the recommended column buffer (in-case the salt concentration was wrong).
Do you guys have any suggestion as to how I should troubleshoot this problem? Any help will be well appreciated.
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
adrian kohsf on Fri May 6 04:05:19 2011 said:
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
Yep 2g of glucose per liter. Thanks for the reply. I think I'm going to try to transform the vector into another cell line.
toshuff on Fri May 6 06:59:40 2011 said:
adrian kohsf on Fri May 6 04:05:19 2011 said:
During your overnight culture, do you grow in LB with glucose? (to avoid leaky expression)
Yep 2g of glucose per liter. Thanks for the reply. I think I'm going to try to transform the vector into another cell line.
Hola, probably you have been done it before without problem, but glucose concentration above of 1g/l could leas at the acetic acid formation by incomplete oxidation, with some risk for the culture viability.About 0.5g/l is enougt to achieve 5-6 OD units in LB. Buena suerte