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CCP4BB  April 2010

CCP4BB April 2010

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Subject:

Re: protein degradation during concentration for crystallization trials

From:

Maia Cherney <[log in to unmask]>

Reply-To:

Maia Cherney <[log in to unmask]>

Date:

Thu, 8 Apr 2010 10:29:14 -0600

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (70 lines)

Hi,

MBP tag:
1.there might be a TEV cleavage site in your MBP variant.
2. your protein needs salt to stay in solution
3. your protein forms aggregates with MBP

GST tag:
you probably concentrate a protease together with your protein. You need 
a protease inhibitor kit to take care of different types of proteases.

It looks like a His tag would be a better option for you.

Maia

vikrant saa wrote:
> Dear all
> I am working on purification of 14 kd protein(pI  8.3, basic protein)  
> that has MBP(maltose binding protein, 45 kd,) tag, and same protein in 
> other vector(pGEX-KT) that has GST tag. During affinity purification 
> in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer 
> throughout the purification.I do on column cleavage with TEV to remove 
> the tag (for crystallization purpose).
> Purification with MBP tag:
>  After cleavage MBP also appear in elution fraction along with cleaved 
> protein of my interest. To remove MBP(pI 5.0) from protein of my 
> interest I have to do anion exchange chr. with DEAE resin(weak anion 
> exchanger) with buffer of pH7.3. Hence I do dialysis against the 
> buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room.
> I have few problems:
> 1) why the MBP elute with protein of my interest ( I tried at low salt 
> concentration also but still elute).
> 2) During dialysis my protein of interest  precipitate.
> 3) If I tried FPLC,  protein of interest and MBP elute in same 
> fraction with superdex 200 column.
>  
> Problem with GST tag purification: it give me impure protein even 
> after enough washes with high salt concentration buffer (upto 2 
> molar). When i concentrate protein in CENTRICON (Millipore, 
> centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer 
> with 5mM BME) it degrade very fast and probably aggregate  as smear 
> obtain on SDS page below the size of protein after concentration (even 
> protease inhibitor not very much helpful) and band intensity of 
> protein of interest almost remain same before and after concentration 
> step.
> Please send me your valuable suggestion to overcome to these 
> difficulty. I have also tried with some additives such as sucrose, 
> glycerol,  PBS buffer.
>  
>  
> With regards
> -- 
> $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
> VIKRANT
> Junior Research fellow
> Cancer Research Institute
> Advanced Centre for Treatment, Research and Education in Cancer (ACTREC),
>  Kharghar, Navi Mumbai, India
> $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
>
> ** 
> **
>  
>  
>  
>
>
> Send free SMS to your Friends on Mobile from your Yahoo! Messenger. 
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