Put the His-tag at C-terminus. Remove rare codons Optimize sequence for translation
You probably got truncations not degradations. Chun -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kn Ly Sent: Thursday, March 19, 2009 4:53 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
