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
> --
> $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
> VIKRANT
> Junior Research fellow
> Cancer Research Institute
> Advanced Centre for Treatment, Research and Education in Cancer (ACTREC),
> Kharghar, Navi Mumbai, India
> $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$
>
> **
> **
>
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