protein solubility problem
satish at a.chem.upenn.edu
Thu Feb 20 16:11:55 EST 1997
In article <malcolm.campbell-2002971551380001 at fibre.plants.ox.ac.uk> malcolm.campbell at plants.ox.ac.uk (Malcolm M. Campbell) writes:
>My questions are these:
>1. Since we want to use the protein for x-ray crystallography, is it
>necessary to conduct purification, etc, under non-denaturing conditions?
No. There are a number of examples of protein which have been purified
from inclusion bodies and then refolded to yield active material capable of
producing diffraction quality crystals.
>2. Is there a method for getting more of the protein out in the soluble
>fraction, or removing it from the insoluble fraction while maintaining it
>in non-denaturing conditions?
Since your construct has a polyhistidine tag, you can solubilize the
pellet in 6 M urea, immoblize the denatured protein on the Ni affinity
resin and refolded it *on* the resin by switching to a buffer without
the chaotrope (the idea is that local immobilization will avert self-
Alternatively, you can try expressing the protein as a maltose binding
protein fusion (some claim that such fusion constructs yield greater
quantities of soluble protein) or grow you overexpressing bacteria at
lower temperatures or co-expressing it with a chaperonin like Gro-EL,
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