Trombin cleavage of fusion proteins

Robert Solomon Bioc rgs at
Tue Sep 6 04:55:18 EST 1994

In article <9409012138.AA65559 at> msseyed at ACS.UCALGARY.CA ("Soheil Seyed Mahmoud") writes:
>Hi there. I have expressed a protein (ca 22kd) as a fusion with
>GST in E.coli. I can purify ther fusion protein on agarose bound
>glutathione beads. However, as I attempt to add the thrombin
>bufer (20mMtris (pH8.3), 5mM CaCl2, with or without 150mMNacl),
>the fusion protein precipitates out. Any suggestions how I can
>get over this problem? Thanmks a bunch.

You have a number of things to try here - the buffer specificty for
thrombin is fairly wide -  Ive cut fusions in 50mM (NH4)2SO4, and in 50mM
Tris pH 7.6, and others Ive forgotten about.  So I suggest as a first
attempt you leave out the calcium (it is *not* necessary) which I suspect
is your problem, and/or change the pH to something a little lower.

Good luck


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