a third way to use the information would be to reclone the part before and/or after the protease cleavage site, i.e. you may have identified (a) stable domain(s) that may crystallize much better than the full-length protein.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij








On 19 May 2015, at 18:48, Fischmann, Thierry wrote:

> The information about of the cleavage site can be used in two ways : possibly identify the protease - as it has already been suggested - or introduce a mutation at the cleavage site which would prevent clipping from occurring.
>  
> The caveat of introducing a mutation is that there is always the possibility that the mutated residue plays a key role.
>  
> Good luck
> Thierry
>  
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Phoebe A. Rice
> Sent: Tuesday, May 19, 2015 12:23 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Protein precipitation
>  
> For one of our more easily-degraded proteins, we added a mix of protease inhibitors (those expensive tablets you can buy) and also put a drop of EDTA into each tube in the fraction collector so that any contaminating metal-dependent proteases would be knocked out as soon as the protein came off the metal affinity column.  That seemed to help.  
> Also, if you can get your protein to stick to the next column in whatever buffer it comes off the IMAC column in, there's no need to dialyze or concentration between columns - the faster you get it clean, the better.
> Good luck!
>  Phoebe Rice
>  
> From: CCP4 bulletin board [[log in to unmask]] on behalf of Michel, Max [[log in to unmask]]
> Sent: Tuesday, May 19, 2015 1:32 AM
> To: [log in to unmask]
> Subject: [ccp4bb] Protein precipitation
> 
> Hi Manjula,
> 
> I had a similar problem: pH, salt, additives and protease inhibitors (I tried them all) didn't work. The protein degraded after lysis of bacteria within 20 min at 24°C to 50 %. I had luck after i cooled down all buffers and materials to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. Finally the Protein was stable for at least one month. 
> You have to take care about temperature induced pH changes. Some buffers change their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to precipitation if you are working close to the pI of your protein.
> 
> Maybe you can try to characterize the protease to find a suitable protease inhibitor. Make some massspectrometry with your degraded sample. An other option is to test via westernblot. If you have some C- oder N-terminal monoclonal well characterized antibody. 
> 
> Greets Max
> Von: CCP4 bulletin board [[log in to unmask]]" im Auftrag von "Manjula Ramu [[log in to unmask]]
> Gesendet: Dienstag, 19. Mai 2015 08:05
> An: [log in to unmask]
> Betreff: Re: [ccp4bb] Protein precipitation
> 
> Thanks all for the suggestions.
> @ Pius,
> I used bacterial expression system.Yes I centrifuged precipitated protein sample and found more amount in soluble form itself, however within a day protein gets degraded. If I have to further purify the protein I have to concentrate the IMAC elutes and concentrate using filters. In this step protein degradation was more and filter used to block and could not complete the process
> .
> Do you use batch method of elution or gradient???
> Also do you add PMSF in the elution buffer???
>  
> Yes Nikhil I did batch elution and included PMSF in all the buffers. and I tried using 500mM NaCl too but no change was observed.
>  
> 
> Thanks and Regards,
> Manjula R 
> Research Scholar
> Department of Biophysics 
> National Institute of Mental Health and Neurosciences
> Bengaluru-29, Karnataka
> INDIA
> E-mail: [log in to unmask]
> Mobile no:+91-9538553356
> http://www.nimhans.kar.nic.in/
>  
> On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN <[log in to unmask]> wrote:
> Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM for IMAC using Ni-NTA. If your protein requires any additional co-factors (like Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.
>  
> On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu <[log in to unmask]> wrote:
> Hi all,
> I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity purification. Eluted with 200mM imidazole.  While elution I could see slight turbid in eluted protein (I get pure protein, single band on SDS-PAGE). However, when I centrifuged I couldn't see any pellet. After some time(a day) protein got degraded. In other method I tried cation exchange purification of lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later degradation.
> After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here also same problem I faced.
> Please suggest me a method where I can get stable protein.
>  
> Thanks and Regards,
> Manjula R 
> Research Scholar
> Department of Biophysics 
> National Institute of Mental Health and Neurosciences
> Bengaluru-29, Karnataka
> INDIA
> E-mail: [log in to unmask]
> Mobile no:+91-9538553356
> http://www.nimhans.kar.nic.in/
> 
> 
>  
> --
> B4U
>  
> 
> 
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