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How to get soluble protein?


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#1 saad

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Posted 30 April 2012 - 12:38 AM

Hi Guys

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.

#2 biozweistein22

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Posted 30 April 2012 - 02:12 AM

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.

#3 saad

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Posted 30 April 2012 - 05:02 AM

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.

Regards,

#4 biozweistein22

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Posted 30 April 2012 - 07:41 AM

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.




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