Extensive washing with 40 or 50 mM Imadazole during His-based purification. And adding Ion-exchange chromatography before gel filtration.

Dear Lubna Alhudhali

Just some questions to better undestand:

1) can you share with us some more information about the purification protocoll?

Eg buffer compositions, volume of the washing buffer used in IMAC.

Sample volume, coloumn and buffer used in gel filtration

I purified many proteins with your strategy 1 and generally the protein eluted in the Flowth Through of the 2nd IMAC (subctractive IMAC) is quite pure since that the most of impurities that bind the coloumn in the 1st IMAC, bind again also in the 2nd IMAC and generally SEC step is not strictily necessary.

2)Did you added DNA-ase in your lysis buffer?

. Probably i'm wrong but since it is a DNA binding protein, maybe that DNA binding to it can alter is properties and in some way induce aspercific binding to coloumn or other proteins that cannot be well separated from it.

best regards

Manuele

Dear Zeyaul Islam Thank you for your suggestion, I washed it with 30 mM one of the protocols I added HiTrap SP HP (Cation Exchange) but couldn't see the difference. Thank you for your suggestion I will try with 50 mM imidazole.

Dear Manuele Martinelli , Thank you for your reply, I have purified different proteins using IMAC with other column as well as with subtractive IMAC, but I have never faced something like this.

I agree with you that the contaminants should be attached to the 2nd subtractive IMAC. Since they were copurified with my protein in the three columns, I think that they are strongly interacting with my protein instead of being attached to the column.

Regarding the purification protocol I basically used two protocols with different buffer compositions, in both protocols the sample volume was around 200 ml, for the washing volume I normally wash the column with 15CV ( almost 150 ml washing buffer) :

Protocol 1 ( HisTrap HP 5ml -->His cleavage --> HisTrap 1ml --> SEC Column (superdex 200 16/60)

• Histrap buffers:

50 mM HEPES 7.5

500 mM NaCl ( was reduced before elution to 250 mM )

30 mM Imidazole

4 mM BME

5% Glycerol

Elution was performed with gradient 300 mM Imidazole.

• SEC buffer:

50 mM HEPES 7.5

250 mM NaCl

1 mM DTT

5% Glycerol

Protocol 2 ( HisTrap HP 5ml -->HiTrap SP --> SEC Column (superdex 200 16/60)

• HisTrap Buffer:

50 mM Potassium phosphate (pH6.5)

10 mM Imidazole

500 mM NaCl ( was reduced before elution to 250 mM )

0.05% NP-40

5 mM BME

10 % Glycero

Elution was performed with gradient 300 mM Imidazole.

• HiTrap HP SP Buffer :

50 mM K-Phos (pH6.5)

50 mM NaCl

0.05% NP-40

1mM EDTA

5 mM BME

10% Glycerol

Elution was performed with gradient 1M NaCl.

I didn't add any DNase during my purification .

Lubna

@lubna You can also try heparin based purification.

The His-tag itself is not really suited for the purest results. Therefore one usually performs an SEC afterward if high levels of purity are required.

What I can also recommend you is not to use His-tag. It is Nickel based (nothing wrong with that).

But Nickel-based beads lie on the High-yield-less purity side even inside the already kinda impure His-tag purifications.

What you can do is use Co-NTA instead. Same protocols, only the Ion is different.

https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/co-nta/

Co-NTA tends to have lower yields than Ni-NTA but noticeably higher levels of purity. That is just the usual trade-off in protein purifications.