How to get soluble protein? - (Apr/30/2012 )
I'm struggling with one of my protein trying to get soluble proteins. I tried lots of different constructs form that protein that predicted to be structured. I used E coli for over expression diff tags, temp, IPTG, buffer, sonication, lysozyme, commercial lysis buffer.... and the results were consistent no soluble protein. I did a couple of refolding exp but no luck to get enough stable and soluble protein.
I've heard about some Ecoli cells competent cells but I don't know if it is just take money and time for nothing. Any ideas and suggestions will be highly appreciated.
Thank you so much for your help.
Please give us some more details of what you have already used used. Bacterial strain ? IPTG concentration and time? Lysis buffer and method? Protein size? If your protein goes to inclusion bodies you can try a mild detergent in the lysis buffer ( e.g. tween-20) if that doesn't work then it gets tricky. You need to defold your protein and the refold it using urea protocols. I can send you one to try but the've never worked for me, couldn't get the right conformation after th refolding.
Thank you for your help.
Yes I tried 18 o/n, 25 o/n and 37 degree for 3-4 hours at 0.1, 0.5 and 1.0 mM IPTG in BL21 (DE3) and Reosetta2.
The size of protein from 20-45KDa +the tag GST, His or MBP.
phosphate, Tris and HEPES, 150-500 mM NaCl, DnaseI, PMSF or protease inhibitor (tablet).
I will try a detergent eventhough I tried 1% TX(100) previously.
If you send me your protocol I can try because mine never worked for me as well.
All my constructs are N-terminal tagged I was wondering if the C-termiual tag could make a big differences.
You have to check if your tagging drives your protein to inclusion bodies, it would be nice if you had the same protein C tagged as a control. Anyway, when I induce o/n with IPTG I have a concentration of 0.05mM and for 2hr inducing 0.3mM. My proposal would be to try o/n at 4C 0.05mM IPTG and add 0.05% tween-20 to the lysis. My lysis buffer consists of tris-NaCl and a Roche table (complete not EDTA free). I will also send you the urea protocol, but you'll have to wait till I get to the lab tomorrow.