Most of the poorly cleavable fusion proteins (usually MBP-TEV) that
I've seen turned out to be solubly aggregated.
ho
UC Berkeley
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Date: Fri, 27 Feb 2009 07:23:43 -0500
From: Stephen Weeks <[log in to unmask]>
Subject: Re: Off topic: Mammalian gene expression in E. coli
Just in case anyone else encounters the same problem, like Artem I=20
have had a few SUMO fusion constructs that have stubbornly refused to=20
cleave even with a 1mg of enzyme (Ratio ~ 1:100) at 37c in the presence=20
of low concentrations of chaotrope. In all cases the problem was solved=20
by inserting a glycine residue between the cleavage site and the first=20
amino acid (always a methionine in my case). This resulted in same=20
constructs being able to be cleaved at 4oC, with a Ratio 1:1000 to=20
1:2000 of hydrolase in 30 minutes (Cleavage was performed in a tyipcal=20
IMAC elution buffer with 250 mM NaCl). Of course by doing this you will=20
no longer have the "Native" amino terminus on you protein but funnily=20
enough have the same additional residue that TEV leaves behind. I=20
perform all my cloning using LIC but if I remember correctly the NYSGC=20
use a BamHI (GGATTC) for cloning downstream of the SUMO cleavage site=20
which will introduce an additional serine residue. I assume, without=20
ever seeing their cleavage data, that they never had a cleavage problem.
In the one case where I finally got crystals of the protein that=20
initially poorly cleaved as a SUMO fusion construct the amino terminus=20
was highly ordered in the structure (I could use the Selenium labeled=20
start methionine for phasing). I'm curious if this can be extrapolated=20
to all poorly cleavable fusion constructs.
Stephen
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