Purifying Iron-dependant recombinant proteins

Liz Gaskell bgyeag at leeds.ac.uk
Wed Sep 27 11:37:20 EST 2006


Hello,

I am trying to express and purify a 6xHis tagged iron-dependant recombinant
protein under native conditions to assay for activity. I am using a Talon-bead
cobalt afffinity resin column. When I do not add any iron to my expression
media I get a lovely pure protein (around 3mg/l) but it is not active. When I
add 100uM Fe2+ I get even more soluble protein that is active, but the iron
seems to be interefering with my purification and I get very little intact
recombinant and a large smear of contaminants when I analyse the elute by
SDS-PAGE.

Can anyone suggest a way to over come this? or a better way of purifying
iron-dependant proteins.

Many thanks

Liz Gaskell



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