recommendations_on_purification

From: Petros Giastas
Date: 26 March 2012 13:04

Dear all,

I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions?

Thanks in advance
Petros

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From: Lorenzo Finci

Petros, 

It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of . Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.

Sincerely, 

lorenzo

Lorenzo Ihsan FInci, Ph.D.

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From: Lorenzo Finci

Etros, 

It has indeed been speculated that high concentrations of Magnesium and/or other metals present in the cell lysate effect the binding of the Histidine-tag, and thus specific conditions for binding and elution need to be optimized for specific elution of your target protein. I believe that the standard recommendations when using a Nickel column to bind to your Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted unspecific binding protein, to try manipulating the binding by lowering the pH of the buffers, and by determining the specific concentration of Imidazole your protein elutes at by using a gradient before progressing to a step-wise elution. Alternatively, you can also try using another metal column such as Cobalt (Talon) with a higher affinity for the Histidine tag.

Sincerely, 

lorenzo

Lorenzo Ihsan FInci, Ph.D.

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From: Carlos Kikuti

90% of the protein could be aggregated and hiding the His tag from the resin. Metal ions might be removed from the starting sample by adding up to 10 mM EDTA in the first buffer exchange cycle, but just after reading "10% of the protein binds" I wouldn't bet much on this. Rather increase the culture volume and move on.

Carlos Kikuti

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From: Artem Evdokimov
Hi Petros,

It's possible but unlikely that your media contains enough metal ions
to matter. More likely options include protein issues like
over glycosylation, proteolysis (removal of the tag) or severe
aggregation. Since you are not experiencing this in BMGY, I wonder why
not to supplement your synthetic medium with yeast extract and/or
peptone and the like?

Artem

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From: Kendall Nettles

I suspect that sometimes the protein chaperones the tag, which is solvent exposed some fraction  of the time.  Try very slow loading or batch binding. 

Kendall Nettles