Hi, The details of the experiment are a bit sketchy. Is this a transmembrane protein or an associated one? What percentage of the structure is expected to associate with the lipid bilayer? Do you know the correct orientation of the protein with respect to the bilayer (i.e. what's inside and what's outside)?
Membrane proteins can be extremely tricky. There are many types of problems that can be relevant here (like abortive translation brought up by Chun Luo), but my best guess is that your protein just isn't being folded correctly and therefore it activates the heat-shock like response (a.k.a. UPR) - causing the kind of proteolysis that you are observing. What this means is that you have to find a way for this protein to fold correctly, which may require you to insert it into the membrane or make sure that membrane approach is established (you did not mention the presence of any kind of membrane-targeting signal, so we have to assume you don't have any in the current construct). You are also facing a strong likelihood that this protein may not work out in E. coli and you have to switch to insect cells or yeast, or mammalian cells in the end. Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kn Ly Sent: Thursday, March 19, 2009 7:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] purification Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien
