Dear Ose,

This is unfortunately a fairly common issue. There are multiple ways to
rationalize what's happening - and even more approaches to reducing the
aggregation issues. It would take too long to list all the options here -
however I would look at the earliest stages of your expression and
purification and try to determine if aggregation is the 'inevitable outcome'
of inherent properties of your protein - or is it the outcome of inadequate
expression, handling during lysis, purification, etc.

Beyond the abovementioned considerations, the following may be useful to
think about: 

a) is the specific activity of your protein close to maximum attainable (or
at least biologically reasonable) value?
b) if you can separate aggregates away from monomers (or proper oligomers) -
does the protein re-aggregate with time or upon concentration?
c) does aggregation state of your protein change with time or during the
course of purification?
d) what is the effect of ionic strength, pH, nature of buffer components,
additives, ligands (substrates?), and so forth?

(a) may be difficult to answer unless you have literature or unpublished
data on this enzyme or its close relatives
(b) is one of the earliest experiments I'd try in the absence of all other
knowledge
(c) should be tracked as you work with the protein as soon as you suspect
that there may be issues
(d) is probably the second experiment I'd try w/o any other knowledge. If
you can use Thermofluor or some other high-throughput method to analyze the
behavior of your protein exposed to various conditions, then you can screen
quite an array of options very quickly

Good luck,

Artem

"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone" 
 Jorge Luis Borges
 
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of ose
toyoyuki
Sent: Wednesday, August 26, 2009 7:55 PM
To: [log in to unmask]
Subject: [ccp4bb] Active aggregates?

 
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
[log in to unmask]

Graduate School of Life Science
Hokkaido University
N21W11 Kita-ku, Sapporo
001-0021 Japan