Purifying Iron-dependant recombinant proteins

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Sun Oct 1 02:42:55 EST 2006


Liz Gaskell wrote:

> 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.

You have four options, but there is no telling in advance which is the
easier one.

First you can separate the protein from excess iron, which will bind to
the His-Tag and block it. Purification must be gentle enough not to
release the bound cofactor, but harsh enough to remove the soluble and
His-bound iron. Ammonium sulfate precipitation of the total protein,
possibly in the presence of His, imidazole or EDTA (check the complex
forming constants at your pH), might achieve that. If you do a
fractionated AS-precipitation you could use this as a first purification
step.

Second, you can purify the apo-protein and try to load it with iron
later. This does not work with all proteins, but since yours is soluble
without iron chances are not too bad. Since the His-tag also binds iron,
it may have to be removed first.

Third, you can forget about the His-tag and use classical protein
purification methods (IEC, SEC, HIC...).

Fourth, you can engineer a different tag onto your protein, that does
not bind metal. 


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