High protein expression but low protein activity - MBP-tagged fusion protein inactive (Aug/21/2005 )

Hello,

I've recently inherited an MBP-tagged (maltose-binding protein) cysteine protease and I have no problems with IPTG induced expression of the enzyme, SDS PAGE shows a high yield, but the enzyme is inactive as shown from fluorogenic substrate assay. I've checked the pH of the buffers, made them fresh, even extracted the plasmid from old stock a retransformed them into fresh competent cells. This used to work in the past. Please any suggestions. Thanks

Do you know how long your protein will last in the presence of ubiquinone? at room temperature? Assuming you work on your proteins in a cold room...

Hi there, in my experience, over expression of certain proteins leads to decrease activity of the target protein. I suspect that the over expression of your protein in the bacteria (bacteria right?), results in mis-folding due to the fact that your proetin is expressed too fast for the bacteria to handle correctly.

To resolve this, try decreasing the amount of IPTG by half and see if activily increases.

HTH

Jeff