Hi Bei,
For the extracellular protein I worked on in graduate school, I
typically purified it from 4 L preps in LB media. The standard
protocol was to do a crude low cut with ammonium sulfate cut followed by
precipitation of the protein with a high cut. The pellet was then
resuspended, dialyzed and loaded on an S-sepharose column.
I experimented with taking the media (after spinning out the cells)
and diluting 1:1 with a low ionic strength buffer and loading directly
onto the S-sepharose column. This worked, but the loading time for
8 L was so long it was not worth it.
Another option might have been to use bulk media to bind the protein
in a batch step. I never tested this.
A final option that we explored was Expanded-Bed Adsorption Chromatography.
This would allow us to get rid of the initial centrifugation step to remove
the cells from the media and would allow fast loading with a high speed pump.
We priced out the media, column and pump from Pharmacia at the time, but
never ended up purchasing the system. The technology looked promising and
should have worked well for our system, but we decided that we did not need
to do too many more preps for the project and just used the standard protocol.
Regards,
Mitch
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of joybeiyang
Sent: Tuesday, April 12, 2011 2:14 PM
To: [log in to unmask]
Subject: [ccp4bb] methods to capture proteins from cell culture medium
Dear all,
My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following:
1. directly load the medium onto a ion exchange column?
2. Amonium sulfate precipitation?
3. anyother thoughts?
Thank you very much in advance!
Best,
Bei
2011-04-12
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joybeiyang
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