HELP: Purification of his-tagged protein
mytelka at mendel.Berkeley.EDU
Wed Nov 20 23:36:41 EST 1996
In article <Pine.ULT.3.91.961121163317.11084B-100000 at sanger.otago.ac.nz>,
David Ackerley <david at sanger.otago.ac.nz> wrote:
>I'm a molecular biologist foolishly trying to enter the realms of protein
>purification, and encountering a few problems along the way. The
>his-tagged protein I'm working with is around 118kDa, and can (although
>not wonderfully clearly) be seen to appear after induction on SDSPAGE of
>crude cell extracts. However, after purification on a Gibco NiNTA resin,
>I am left with barely detectable amounts of my protein, not nearly enough
>for sequencing to confirm id. The only product I am managing to purify
>significant quantities of is the 70ish kDa E. coli GRO-EL protein (or
>something like that?) that is apparently commonly copurified in this type
>of procedure. I have tried the following protease inhibitors, both
>separately and together; PMSF, pefabloc, pepstatin, TPCK, and soybean TI.
>But no luck. Sigh...
> Anyhow, my question is this: has anyone out there encountered
>similar problems, and if so, what did you do about it? I am considering
>looking at other purification matrices, but thought I would see if anyone
>with more experience than myself (ie just about anyone) had any helpful
>(and hopefully cheaper!) suggestions to make. Any such suggestions will
> Cheers, David Ackerley
I suggest that you try to find out what the problem is before you
try to solve it. Proteases can be the problem, but they aren't the
only ones. Here are a few thoughts:
1) Expression system. A good expression system should give you a
wopping band, not a barely detectable one. Try optimizing
overexpression conditions (time, concentration of inducer). The more
you start with, the easier it is.
2) Lysis. Are you losing a lot in the lysis or in the spin to remove
insolubles? Is there an appearance of a smaller band (or a smear)
that might indicate proteolysis? If a lot is insoluble, you might
try resolubilizing the pellet, either in a harsh denaturant (like
guanidinium) or with something like sarkosyl (there's a Burgess
paper on omega protein purification that talks about this and it
has worked well for me on occassion).
3) Column. Is it binding? Is it releasing? Everything that goes
on has to go somewhere, so check all of your fractions. If it flows
through, either you're doing something wrong or the tag you're
using is poorly positioned (for example, if the place you put the
tag turns out to be buried, it won't work very well). If it
isn't eluting, there is probably something wrong with one of
your buffers, or you aren't using a full range of elution conditions.
4) Gel. Depending on concentations of the load and whether you
elute with bumps or a gradient, your final sample may end up being
fairly dilute. Are you loading enough to EXPECT to see anything?
If you're precipitating before loading, are you resolubilizing
properly before loading the gel (I had a problem with this when
I first started doing this--saw nothing on my gels until I
added more fresh BME to my load buffer).
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