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.
Download Now! http://messenger.yahoo.com/download.php