Dr. Peter Gegenheimer
PGegen at UKans.edu
Fri Aug 7 18:57:32 EST 1998
On Mon, 3 Aug 1998 09:29:49, "Lars Komorowski"
<larskomo at physik.mu-luebeck.de> wrote:
> I am trying to overexpress a 17kDa-Protein in E.coli BL21 with Novagens
> pET24a Vector. I inocculate my LB medium from an overnight culture, let the
> culture grow until the OD reaches 0.6 and induce with 1 mM IPTG. After one
> night there is a band at 25 kDa in SDS-PAGE made with total cell protein.
> When I try to separate cytosolic proteins from the rest and make another
> SDS-PAGE with both fractions there is no band there.
An another poster answered, it is common that overexpressed proteins
will aggregate into insoluble "inclusion bodies". If you were really
just examining the soluble cell fraction by SDS-PAGE, examine the
cell debris. (If the protein is there, the cell debris ought to be a
big white pellet.) The lesson from this is that when you think
you've lost something, make sure you've accounted for 100% of the
places where it could be--no matter how unlikely.
If the protein is in the pellet, you can get a reasonable
purification by washing the pellet to remove trapped cytoplasmic
protein. Homogenize the pellet (in tris/edta/NaCl or whatever you're
using for cell lysis) and spin it back down; repeat 1-2X.] Addition
of Triton or sarkosyl would help remove membrane proteins.
Inclusion proteins are formed when nascent polypeptides don't fold
completely, and the exposed "interior" regions, which are usually
hydrophobic, of only polypeptide associate with adjacent polypeptide
chains rather than with their "own" chain.
To avoid inclusion body formation, two methods which have been
well-documented in the literature to work _sometimes_ are: 1) Induce
your target gene to a very low-level; use 10-100 micromolar IPTG and
let the cells incubate (plenty of aeration!) for a day or more. 2)
Grow the cells below room temperature--15 to 19C--just prior to and
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