Protein Expression

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Tue Feb 18 17:28:51 EST 1997


In article <3309EBD3.E87 at urz.uni-heidelberg.de>,
   Klaus Lun <klaus.lun at urz.uni-heidelberg.de> wrote:
->Im trying to express a transcription factor in bacterial cells, and i am 
->using quiagen's pQE-30 vector with an N-terminal 6*His-tag and also 
->quiagens Ni-NTA resin to purify the protein. The expression seems to 
->work well, i got a specific protein that binds to the Ni resin (under 
->native conditions !), but when i want to perform a large scale 
->isolation, lots od other proteins also eluate from the column after 
->washing.
->So, how can i get rid of this proteins ? the manual say trying washing 
->with 1% TX-100, 1M NaCl, 40 mM Imidazole, 30 % Glycerol, 10 mM 
->ß-Mercaptoethanol .. ?? 

	Nacl might help but unlikely. TX-100 - very probably (YMMV). Glycerol
and bME are useless in this case, IMO. 

Most likely the source of contamination is "true" IMAC binding. Then the only way
is 1) carefully optimize binding/washing/elution conditions and/or 2) cleanup with 
another chromatography after IMAC. 

->The washing is performed in wash-buffer (10% 
->Glycerol, 0.3 M NaCl, 50 mM Na-P pH5.8).
->Should I now add all these components to the wash buffer at the same 
->time and try a big mix, or test them seperately ?? Can some of the 
->reagents affect the activty of the protein ?? Has anybody made 
->experience with this reagents ? I need the protein later for DNA 
->bandshift assays, so it's native function is essential. 
->
->I elute the protein with an 0-0.5 Imidazole gradient in wash-buffer.
                                                          ^^^^^^^^^^^
Imidazole gradient at pH 5.8? Most of it's molecules are protonated
at this pH... (pKa 7.0)  

Here is how I go about IMAC purification:
1. Don't bother with low pHs. Use some buffer with pH 7-8 and NaCl 
of > 300 mM. In small-scale exp., bind everything at
O mM imidazole and elute with ~ 3 column volumes steps of 50, 100, 200, 500 mM
imidazole. Find out where your protein elutes. 
2. In small-scale exp., make sure your protein binds to the resin in the
presence of 3-4-fold lower imidazole concentration. 
3. Use this imidazole conc. in binding and washing buffers. 
4. Wash until very little protein is coming off (this is where all additives
like higher salt or detergents can really help - by increasing stringency of
elution conditions for the pool of "tailing" proteins. On the other hand, neither
detergents, not high salt negatively affect binding in IMAC so they can be safely
included in binding buffer as well). 
5. Elute with at least 10 column volumes linear gradient ending at 2-3 fold higher
then "eluting" imidazole concentration. 

In my experience, such procedure results in as clean prep as it could be. 


-> How 
->long can the factor be kept in this buffer at 4°C ?? Can I freeze it at 
->-20°C ? In which conditions can I do a long-tern storage of the active 
->protein ??  And do i have to get rid of the Imidazole in the elution 
->buffer ?? Does it interfere with the binding assay ?? and if yes how can 
->i get rid of the imidazole ??

All of the above depends on your particular protein/assay and has to be determined 
empirically...


- Dima


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     / /\         Dima Klenchin
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   / / /\ \       klenchin at facstaff.wisc.edu
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