protein/enzyme activity - lost activity after his tagging (Sep/19/2005 )
i'm working with a bacterial enzyme, when i purify this enzyme, it has high activity in crude lysate and after first column but loses it gradually afterwards, to get it 90% pure, i must use at least two columns with a amm. sulfate step before loading onto first column. so then i decided to put a His tag on both N- and C- terminal, but while C-term has no activity ( not even the crude lysate) , N-term has no expression. i'll appreciate if somebody could help on this, loss of activity and expression.
PS: please don't give any suggestions of cloning part, everything is fine there ( sequence, frame etc.).
Have you looked at the NEB Impact-CN system? It uses a fusion of a C or N terminal chitin binding domain, followed by binding to chitin beads. The cloned part of the fused protein is then cleaved off of the chitin beads with DTT, eluting the native form of the protein. I don't know how difficult your protein might be to renature, but this might let you purify it to native primary sequence in a single step.