How do I lyse these cells?

CAS cs_nanae at acornhosting.net
Tue Mar 1 16:20:35 EST 2005


Hello all,

I've always had a sonicator handy, and now I find myself without one.

So I'm looking for suggestions as to how to get an over-expressed
soluble enzyme out of some E. coli BL21(DE3) cells.  No sonicator, no
french press.

I've looked at the CLONTECH kit to use with the TALON his-tag system,
but it's expensive and my budget is tiny.  (Otherwise, I'd just buy a
sonicator!)

I'm going to try freeze-thaw cycles, which worked very well with
protein G (57aa) in BL21(DE3), but I'd appreciate other suggestions for
if that doesn't work.  (Note: the freeze-thaw cycles do not cause cell
lysis (provided it isn't BL21DE3's with pLys), but they do let the
protein leak out, resulting in a pretty clean prep without a lot of
cellular garbage - if your protein will come out of the cell under
these conditions, I highly recommend it.)  I guess I'm skeptical that
freeze-thaw is going to work with this 250 aa enzyme, so I'd appreciate
other ideas to try in case it doesn't.

Thanks!
--Cat




More information about the Methods mailing list