Good evening,

I work on aminopeptidase A, a 160 kDa membrane enzyme monozinc tagged with a polyhistidine.

I am trying to purify my enzyme but I have poor results and the elution is not good enough.

My protocol : I harvest 12 falcon 225cm2 of CHO cells which stably express my enzyme. I make a membrane preparation and I solubilize my membrane in TrisHCl 20 mM pH 8, 1% Nonidet P40 (NP40) overnight at 4°C. After that I ultracentrifugate my sample and I collect the supernatant.

Meantime I prepare my Talon Cobalt Affinity Resin from Clonetech by equilibrating it with my solubilization buffer. Then I let to bind the supernatant with the resin overnight at 4°C. Finally I put into column my mix. I collect the flow through then I wash my resin two times with solubization buffer, then I make a two step elution with first TrisHCl 20 mM pH8 100 mM NaCl 10 mM Imidazole 0,1% NP40 and in second TrisHCl 20 mM pH8 100 mM NaCl 250 mM Imidazole 0,1% NP40. Finally I change my buffer to TrisHCl 50 mM pH 7,5 100 mM NaCl 0,1% NP40 with Amicon Ultra-15 100kDa filter.

This protocol resulting in 40% recovery, a 10 time yield and 50% loss in flowthrough and first imidazole Wash. And in Ironnitrate gel I a have a thin strip at the size of my protein and many strips under 100kDa wich is problematic.

So I want to know if someone can give me some advices to improve the recovery and to use correctly my amicon.

Many thanks in advance =)

Pierre