about using BL21(DE3)plys - (Aug/16/2005 )
I used the strain BL21 pLys for overexpressing protein. I collected cells and keep them at -80C. I took them out today from the freezer and thawed on ice. But, when I suspended the cells with lysis buffer for sonication, I felt that cells were very sticky and viscous. So, I could not suspend very well. Anyway, I sonicated the cells and load on to the Ni-column but lysate clogged the column. I found the lysate itself is also very viscous. I could not purify at all. I do not what the problem and I am very desperate. Could you tell me what to do to solve this problem please?
i too had used BL21 for expression and purification of proteins..ofcourse i had not frozen the pelleted cells..i try to use them immediately for lysis..but still,the resuspended cells are viscous,but not the lysate..one thing i could be of help to you is that,i too had tried sonication to disrupt the cells..but that does not work well..you can try to lyse the cells using FRENCH PRESS!..this is very efficient..
the next point is,how do u clear the lysate before you apply to the column?? you have to standardize the protocol for your protein..you can use ultra centrifuge or sometimes centrifugation at 15000 rpm for 10 minutes at 4degrees works well..
Maybe you used too small amount of buffer for resuspension.
I usually apply 50-100ml buffer to the E.coli pellet from 1L(OD=3-4), and that's enough.
If you need to decrease the volumn, apply DNase before column application, that'll help.
Then, do I have to use sonicator or french press for cell lysis?
i literally just finished two protein preps with pLysS strain (like 5 minutes ago), starting with FROZEN pellets. i thaw it out on ice and then incubate on ice with about 5 ml lysis buffer per 250 ml bacterial pellet and 0.5-1 ml freshly prepared 1 mg/ml lysozyme for 30 mins. you can use more lysis buffer if you need, but that just means more work in the french press which is a literally painful process. i do three french press passes on ice. and then, because i'm anal and very slightly obsessive-compulsive, i divide the now homogenous lysate into 2 ml batches and sonicate the f*** out of it at highest output ON ICE three times for 15 seconds each. you then ultracentrifuge the lysates for about 15-20 minutes at 20000-30000g, depending on your protocol. keep the pellet aside (don't throw it out cos you may want to check if your protein is in there if it isn't in the supernatant), and combine the supernatants to which you'll add your purification resin. if your protocol calls for a washing step, i recommend doing that on the column itself to minimize busy work, as well as resin loss. if you don't want all the work in the lysis step, i'd definitely go with the french press over sonication, always making sure i have enough lysis buffer. a good way too know if your lysis in the french press is working is that the dirty brown color gets lighter-- a milky, off-white or white thin liquid consistency is what you're aiming for. i understand this process might be kinda slightly redundant but it only takes a little more time than usual and you can always bed your protein in the column overnight before elution... i've purified different proteins throughout my career and never never had a problem with viscosity and i get nice clean globs of my protein best of luck!