I'm currently working on the purification of EPO which is secreted from CHO cell . for final step of purification I have to applied IMAC-Fe(III) for an especial goal.(Endotoxin removal)
could any one help me regarding below question?
I want to apply human EPO on IMAC-Fe(III) in this base buffer: 0.5M NaCL, 0.02Maccetate, in pH 5.5,
Dose EPO capture to this resin with Fe ligand in this buffer base?
for elution I want to applied phosphate buffer, to remove endotoxin from protein. First of all it is necessary for me to know EPO with its amino acid sequence and this buffer, could capture to the ligand.
If this is a His-tagged EPO variant, or if EPO is known to bind IMAC resins via surface His patches then yes, it is not unreasonable to expect it to bind.
There are two histidyl residues on the surface of epo. I would use regular nickel ions. If you want to use iron ions I would not use phosphate since it has high affinity for iron ions.
Dear Gonzalez
As I said before, Im going to use phosphate in elution buffer to detach epo protein from iron ion,
this is my question, does epo with two histidyl residue have any affinity to iron ioin this buffer, 0.5M NaCl, 0.02M acetate pH 5.5?
Dear martin
Epo has two histidyl residuein its sequence, with out his tag in its sequence
I was wondering is it possible these histdiyl group
It could be, given the proximity of the histidyl residues. As I said before why aren't you using nickel? I am pretty sure it will bind to nickel.
I want to apply iron ion to remove endotoxin, lipid A in endotoxin has phosphate group and has good affinity to iron ion, by manipulation pH I want to remove lipid A from protein solution
Hi there,
As Histidine side chain is vastly protonated at pH5.5 it shouldn,t exhibit any affinity for the matrix.
Dear Dominique
could I ask you recommend a proper pH value regarding this process?
this is my Idea, I want to apply EPO on IMAC Fe resin in pH 5.5 to capture almost all of protein solution to Fe ion and find endotoxin in flow through. I know that endotoxin has high tendency to adsorb Fe in pH 3.5. So by apply pH 5.5 I can create a condition to capture protein in Fe along with lowest capture endotoxin to ligand. By manipulating of pH in elution step and decrease it to 3.5 I want to separate EPO from endotoxin.( maybe some endotoxin will capture to ligand in latest step)
Could I ask you recommend me a pH for apply EPO on Fe- IMAC? this is the buffer for loading EPO on resin 0.5M NaCL, 0.02Maccetate, in pH 5.5
Thanks.
Hadi,
You need a pH of at least 7, preferably 7.5-8 to guarantee that the His residues can form part of the metal chelate.
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