My enzyme is not active :( - (Mar/04/2008 )
Hi Bioforumers,
I've been having problems in trying to obtain activity with my enzyme. Funny thing is that a few months ago it was active and now it doesn't seem to work! The protein is around 70kDa and inserted into a PET30a vector.
When it was active I performed expression as follows: Expressed in BL21 DE3 Ecoli (400mL) and induced with IPTG to a final conc of 1mM - BTW expression took place at 30C for 3 hours. Then sonicated and purified the protein using Ni2+ chelating. Did the activity test and ta dah - IT WAS WORKING! 
Now I come back to it after a few months and it doesn't seem to work. I tried lower induction temps (16C, 26C), expression in 2 Litre volumes, protease inhibitors whilst sonication, higher IPTG cons (5mM final con), unfolding and refolding the protein with GSH and GSSG but nothings works. 
I've ran SDS gels + Western blots and can see some expression (though it's not too high) - I just can't figure out what the problem is.
Does anyone have any suggestions?
Thanks a lot!
Is your problem one of expression, or is it that your well-expressed, well-purified enzyme no longer works?
Have you resequenced your construct? Have you done a time-course of OD through the induction phase?
Have you resequenced your construct? Have you done a time-course of OD through the induction phase?
It's an expression problem. I did the usual transformation into BL21 (DE3) and then expressed following my "working" protocol. I haven't resequenced the construct.
I checked my FPLC chromatogram and the active enzyme had a max. UV of ~200mAU. This was when I expressed in 400mL. Now it has a max. UV of ~80mAU (from 400mL). When I express in 2L, I just about manage to get 150mAU.
I'm sure something's gone wrong with the expression
Thanks for your reply
Did you grow your bacteria from glycerol stock or re-transform? Have had some problems with BL21 Codon+ RIL when I grow from glycerol stocks. Usually the problem is gone when re-transforming bacteria.
Good luck!