Just try crystallizing it. What is a crystal but a "massive
aggregate"? That they are still soluble and active is great news.
As a grad student, I had a similar phenomenon with an early project. I
showed a gel in group meeting where both activity and aggregation were
obvious, said the aggregate was no problem, got ridiculed when I said
I was going to throw it in trays despite what anyone said, had giant
crystals after a few trays, and solved the structure with miras.
Get the structure and then worry about why it's aggregating. The
structure will probably provide you with the clues you need.
On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote:
Dear all,
This is a question on how to cope with the protein that seems to form
massive aggregates in solution but enzymatically active.
I'm working on a protein whose molecular weight is around 70kDa and
can be
divided into two domains (say A and B domains). We expressed this
protein in
E.coli fused with GST and purified using some chromatography. The GST
affinity chromatography works well and proteinase digestion to
remove the
tag does wonders too. The purified protein was confirmed to be active
enough, we can detect both activities from these two domains. But the
retention time from the gel filtration clearly shows it is awfully
aggregated (comes out at the void region). DLS measurement indicates
the
averaged diameter is around 45 nm, which I feel is a bit too long.
Analytical ultracentrifuge result implies that the distribution of the
molecular species is wide, some portion got precipitated with 1K rcf
(means
the molecular weight is more than 5MDa) and the rest is ranging from
1MD to
5MDa with a peak at 1MDa.
I made new two constructs covering the A and B domains respectively,
both of
them are active again, but only the A domain has got the same
symptom as the
intact protein. The B domain seems to exist as a monomer in solution.
Here come my questions, (I) How can I interpret this phenomenon?
(II) Is
there anything we can try to change the situation? (III) Does it
make sense
to try crystallization? (probably not).(IV) Has anyone got such
experience?
I tried the methylation on lysine side chains, I also tried the
buffer with
0.2M arginine or 10% glycerol but the all the results just seem
hopeless.
The protein before the proteinase treatment also comes out at the void
region from the gel filtration.
cheers
toyoyuki
--?
Toyoyuki Ose
o...@sci.hokudai.ac.jp
Graduate School of Life Science
Hokkaido University
N21W11 Kita-ku, Sapporo
001-0021 Japan
