Protein unavailable for IMAC binding

[Analia Alet]

Once I had a problem like this with BL21(DE3) trying to express a protein with 
a 6xHis Tag. And I tried induction with lactose to allow protein to folder 
properly. It takes much more time, but it could work. Another thing to do 
could be to make the culture in M9 supplemented with casein-aminoacids (CAA)1% 
P/V so you have under control the "food" the strain is "eating" (i don´t 
remeber the properly words in english)

Hope it works
Best regards 
Analía


DK wrote:
> 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 
<factors.

<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 
<with.

> 
> 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 
>> crystallisation. 
> 
> 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. 
> 
> DK