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[ssubedi]

Hi,
I recently am working on a mutant protein (2 nucleotides mutated resulting W into F). The construct before putting the mutation was doing great but now protein has started going into inclusion bodies post IPTG induction-lysis-sonication-centrifugation. I am using BL21 bacteria which is some 8-9 years old. I suspect either the bacteria or the mutation that has caused the protein to stabilize or denature. Has anyone run on something like this? Any suggestions?

Thank you.

[biozweistein22]

Hello friend, I have experienced the same problem when purifying a mutant protein. In my case, same as in yours, the wt protein expressed good. Try 0,05mM IPTG overnight at 15-20C to allow the protein to fold properly and also add a mild detergent to your lysis buffer ( I used tween 20, 0.05%) for your inclusion bodies problem. I also use both french press and sonication to lyse the bacteria, if you have a french press i suggest you use it before sonication, if not sonicate a bit more. Get back to me for any questions you might have. I hope this solves your problem