Dear all,
I am using BL21(DE3)pLysS to express my protein (as indicated by the red arrow). Both pictures showed significant protein expression pre- and post- induction. The only difference between these two pictures are the competent cell batches - both prepared by CaCl2 method. However, proteins from the 1st competent cell batch (1st pic) was mostly in the soluble fraction whereas proteins in the 2nd batch (2nd pic) was mostly in the pellet.
What could be the cause of this problem? I would appreciate any answers/suggestions.
Thanks in advance.
(M = marker, L = lysate, P = pellet, S = supernatant, FT = flow-through, W = wash, and E = elution fractions).
Hi there,
Transformation method has no effect on the solubility of expressed protein as its role is only to introduce the plasmid into the cell. What you have to check is actually the culture and induction protocol you have followed. If the protein behaves differently, something has got to have changed in between (strain, culture medium (composition and concentration of antibiotics), T°, OD at induction, concentration of inductor, length of induction, composition of the lysis buffer, extraction method...).
Hi there,
Transformation method has no effect on the solubility of expressed protein as its role is only to introduce the plasmid into the cell. What you have to check is actually the culture and induction protocol you have followed. If the protein behaves differently, something has got to have changed in between (strain, culture medium (composition and concentration of antibiotics), T°, OD at induction, concentration of inductor, length of induction, composition of the lysis buffer, extraction method...).
I agree with Dominique. The competent cell batch shouldn't matter (as long as it's the exact same strain, etc.). I'd go back to your expression and make sure everything was done as similarly as you think (all the variables mentioned above, including using baffled flasks, shake speed, etc.). OD at induction can make a big difference even when a researcher subjectively deems it "close enough." If all else fails, just run it all again and double check.
Good luck!
I totaly agree with both answers. I know that the protocol of Bl21(D3)pLysS sets that you should transform each time you produce protein but it is not always the rule. You can try to freeze your Bl21(D3)pLysS in grow media with antibiotic (for plasmid and Bl21) and glyserol (15%). I tryed -70°C and liquid nitrogen and in some cases one works beter than the other. You just need to grow the transformed bacteria in agar media before over night culture
Good luck
Hi,
I would argue against freezing glycerol stocks of transformed expression B strains as they are not recombinant disabled as are the common cloning strains (DH5alpah etc.). This might work once, twice or more depending on your plasmid and gene but there is a good chance it won't be stable and finally the B cells will edit (out) a problematic/toxic gene.
I recommend fresh B strain transformations to be used within a week, sorry that this does not answer your question but it is an important point to make.
Solubility should not be affected, it has to be another parameter as already suggested.
Thank you very much for all the suggestions. I would go and recheck the protocol very carefully.
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