metal chelation for proteins (View forum version)


Posted 17 October 2012 - 06:22 PM

I am dealing with a metallo-enzyme. The idea is to chelate out the metals in it and try to replace it with other divalent metals to see if that improves activity. It is known that the protein needs 2 equivalents of metal to be catalytically most efficient. I have been growing the cells by adding the chelator at OD 0.2-0.3 and then adding the metal and IPTG at around OD 0.6. I am however, unable to ensure an uptake of 2 equivalents of metal by the protein (as evident by ICP-MS data) after an 18 hour growth. Is there any specific protocol that I need to follow to rectify my problem? Thanks.


Posted 17 October 2012 - 07:12 PM

It seems unlikely that this will work with whole cells. An alternative would be to lyse the cells and chelate metals from the lysate, or to purify the enzyme and replace the metal in the purified enzyme.


Posted 17 October 2012 - 11:19 PM

Selecting media lacking completely or very negligible presence of metal to be replaced. adding metal to be replaced at start may be options.


Posted 22 October 2012 - 11:02 AM

Thank you for the response. I will try to chelate the metals out of the purified enzyme and try adding different ones.