[Protein-analysis] MBP fusion protein purification & aggregation

jayhoony from hotmail.com via proteins%40net.bio.net (by jayhoony At gmail.com)
Fri Feb 2 13:49:55 EST 2007


Hello people out there.  I have been banging my head to the wall for
the last 6 months because my protein purfication/aggregation problem,
and I was wondering if any of you could help me out here.

I am trying to express a human protein fusion with MBP in pMAL-c2x
vector.  (this protein is known to be insoluble in bacterial system,
so I used MBP as a solublizer).

I can express the fusion protein at good levels with good soluble
fraction, and I can get fairly pure protein (based on SDS-PAGE) thru
batch or small column purification method.

However, I have had a problem with cleavage of MBP with various
concentration of protease.  (I tried different proteases, and
different linker length...etc).

It turned out that when I ran the purified sample of fusion protein in
the gel filteration column, most of my fusion proteins were in
aggregated form.  I have tried different salt concentration and pH,
but haven't been able to sort out this problem of aggregation after or
during the purifcation step.

Is there any way to prevent the aggregation of fusion protein?

Is there a chance that my protein did not even fold at all, but since
MBP is pretty big, it came out in soluble fraction?

I would appreciate any comments or suggestion here.

thanks in advance.



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