You can try using affinity tags on both the N- and C-termini of the protein, eg. MBP on N and His on C.
ho > Date: Thu, 19 Mar 2009 23:53:14 +0000 > From: Kn Ly <kn...@auckland.ac.nz> > Subject: purification > > Hello everyone, > > I am expressing a 100 KDa eukaryotic membrane protein in E coli. The prot= > ein > is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KD= > a. > > 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
