Protein unavailable for IMAC binding
(by hzhen from freeuk.com)
Fri Feb 29 15:00:43 EST 2008
> In article <47c736ea$1 from clarion.carno.net.au>, Bean Long <ben.long from yourfinger.anu.edu.au> wrote:
>> Hi all,
>> I'm expressing several proteins in BL21(DE3) using 0.5 mM IPTG for
>> induction. One protein is proving a little difficult in that it is
>> successfully extracted into the soluble fraction but only a relatively
>> small proportion of this is subsequently bound to IMAC.
>> I say relatively
>> small in that a huge amount does not bind to IMAC but recovery of pure
>> protein is still quite good. It would seem that a large amount of the
>> protein is forming soluble, yet unbindable (occluded tag), aggregates.
> Absolutely correct. This kind of things happens all the time with
> MBP fusions (MBP is so soluble that it keeps any garbage in
> solution and from forming inclusion bodies).
True, but I think the OP meant His-tag.
>> I wondered if using lower IPTG concentrations would reduce the total
>> protein concentration and therefore the likelihood of aggregation or is
>> there another approach?
Yes, it may. Although it is not the reduction of total protein
concentration that helps, it is the rate of protein synthesis - the
protein needs time to fold properly, and partially folded protein tends
to aggregate, lowering the rate therefore reduces the amount of
partially folded protein present at any one time, hence less likely to
aggregate. You need to experiment with the amount (I have use as low as
0.02) and that is dependent on what kind of cell and plasmid you use
(whether it included extra lacI or lacIq). However, it can be very hard
to get some proteins to solubilise properly and that may be due to other
Other (perhaps better) possibility you may consider to reduce the rate
of synthesis include lowering the temperature (down to 25, 20, 15 or may
be lower, only you can determine that by experimentation. I think I
have use 4 deg before.) after induction and left overnight. You can
also consider folding modulators but that is really not necessarily (and
perhaps too much work) when you have sufficient folded protein to work
> Before that, characterize fraction that does elute. In numerous cases
> here, the bound/eluted fraction was still an aggregate that precipitated
> immediately after MBP cleavage with rTEV.
>> I do not wish to use denaturing agents (e.g.
>> urea or guanidine) during purification as the protein is bound for
> You could still try 0.5-1.0 urea, which is not denaturing for a vast
> majority of proteins but sometimes disrupts aggregates very efficiently.
More information about the Methods