MBP Fusion Proteins - Some Difficulties

Michael S Curtis (Michael_S_Curtis@ccmail.bms.com)
Tue, 26 May 1998 22:03:34 -0400

Hello.

I am currently working with some MBP fusion proteins (pMAL) and I am
trying to Factor Xa cut them to release the fusion partners. I am
having two difficulties:

1. I am using Amylose resin from NEB to purify the proteins and the
capacity seems poor. I am getting protein on the resin however the
majority remains in the cell supernatant. Increasing the amount of
resin does not seem to help. Has anyone seen low capacity with this
Amylose resin? And if so are there any remedies?

2. I am doing the Factor Xa cut at pH 8.0 with the fusion still bound
to the beads. The cutting seems to be going well, but I am getting
allot of MBP in the prep. I have filtered the cut supernatant through
a 0.22um filter thinking that there may be resin in the prep but the
MBP is still there. Has anyone seen this phenomenon and is there any
solution short of a second purification?

This is my first experience with MBP fusions; this fact could explain
everything.

Any advice would be appreciated.

Thanks.

Michael Curtis
Bristol-Myers Squibb
Biotechnology Development Laboratories
Syracuse, NY
Michael_S_Curtis@ccmail.bms.com