Most of the poorly cleavable fusion proteins (usually MBP-TEV) that I've seen turned out to be solubly aggregated.
ho UC Berkeley ------------------------------ Date: Fri, 27 Feb 2009 07:23:43 -0500 From: Stephen Weeks <[email protected]> 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
