I am using E. coli BL21 (DE3) to express proteins but I am getting problem with lysis, sometimes it work and sometimes it doesn't.
I am using lysis buffer (50mM NaHPO4, 30mM NaCl, 0.5 Triton-X, pH 8.0) plus 100 mg lysozyme, protease inheritors and DNase. After resuspension I freeze the mixture at -80 C overnight and then sonication for 3 minutes in intervals
unfortunately I can't get proper lysis and many times the protein comes off as insoluble.
Does anyone have a solution please?
Hello Ammar,
It looks like your recombinant protein is produced as insoluble inclusion bodies. Did you try to look for E. coli soluble proteins in your crude extract ?
Hello Ammar,
Solubility of protein and lysis greatly influences the type of detergent used. Try replacing tritonX with Octyl POE or LDAO.
Dear Pierre Béguin and Rohan Shah
I am sure that the protein is soluble and we expressed it many times before
Dear Ammar
If you are sure that the protein is going to be soluble, then probably the problem is lysis of the cells. Sonication is not as effective, so i ll suggest you to lyse the cells by french press, and you can even check the lysis efficiency post sonication or french press via a microscope to check if the cells were successfully lysed. The refolding is always a bit tricky part. So you can try different detergents as Rohan suggested.
Best
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