Very insoluble expressed protein

Neil Saunders saunders at bio.vu.nl
Wed Aug 12 09:56:23 EST 1998


Dear all,

I wonder if anyone has experience with incredibly insoluble proteins
expressed from a pET vector?  My protein is about 14 kDa size and has 4
cysteines, 2 or more of which may be involved in metal binding. After
several weeks of optimising induction conditions (insert in pET22b,
BL21DE3), I finally have a dark brown colour in my cell pellet; on
lysing the cells, this colour is pelleted by a low-speed spin,
indicating aggregation.
The problem now is, it will not re-dissolve! I know this is a common
problem and have researched a number of protocols; currently I'm trying
to redissolve in 50 mM Tris pH 10.0, 8M urea, 20% glycerol, 10 mM MgCl2
, 10 mM DTT, 1 mM EDTA, 0.2M KCl, and the pellet is completely
insoluble, after incubation at room temp. for 1 hour. It's got to the
stage that I'm beginning to wonder if it's even protein!
I'd be very grateful to hear from anyone with similar experiences/ideas.

Neil Saunders

Department of Molecular Cell Physiology,
Faculty of Biology,
Vrije Universiteit,
De Boelelaan 1087,
1081 HV, Amsterdam
The Netherlands

phone: +31 20 4447194
email: saunders at bio.vu.nl
WWW: http://www.bioch.ox.ac.uk/~nfws




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