Hi all,
I am purifying a basic protein with pi 7.8. I used 50mM tris 8.8, 300 mm NaCl, 5% glycerol, 0.1mM PMSFand 5mM beta ME in the buffer and performed affinity purification. Eluted with 200mM imidazole. While elution I could see slight turbid in eluted protein. However, when I centrifuged I couldn't see any pellet. After some time(a day) protein got degraded. In other method I tried cation exchange purification of lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later degradation.
Please suggest me a method where I can get stable protein.
Manjula,
Are you attempting to purify a protease? If not it looks like that in both methods that you are co-purifying a protease. I would suggest adding the usual protease inhibitor like PMSF (serine protease), EDTA (metalloprotease, this should be added after the IMAC step) or N-ethylmaleimide (cysteine protease, but be carefull this may affect your protein since it is a non-specific cysteine alkylating agent).
If you are attempting to purify a protease I hope that you expressed the proenzyme. Purification of the proenzyme should be done and only after it is purified then you gently activate it.
Hope this helps,
Marc
Hi Manjula,, as u have mentioned, u had eluted ur protein at 200 mM Imidazole, so I presume u r using Ni-NTA IMAC Chromatography.
Ni-NTA resin cannot resist 50 mM BME, to my knowledge only Qiagen provides highly resistive resin which can withstand only up to 10 mM BME. I personally never use more than 1mM BME when using NI-NTA resin. If BME is must for ur protein, u can always add after eluting from the column or during buffer exchange step.
So next time completely avoid BME and u will see there will be no turbidty after u eluted ur protein. All the best!!
Sorry by mistake I have written 50mM beta ME, I used 5mM only. And I included 0.1mM PMSF.
Hi Manjula,
As Bhanu pointed out, a high concentration of BME is not a good idea with Ni-NTA and 5 mM is a little bit high. I prefer not to go over 3 mM, as high BMA concentrations will strip Ni2+ ions from the resin and the free floating ions can lead to aggregation of the protein molecules via their his-tags.
Ni-NTA is a good first step purification method, but in most cases it will not give you pure protein on its own. If you run your eluted protein on an SDS-PAGE gel and you see several extra bands, one or more of them might very well correspond to a protease, which might explain why your protein degrades. I would suggest you add protease inhibitors (we use Roche EDTA-free protease inhibitor tablets) to your cell lysate and to follow your Ni-NTA with a size exclusion chromatography step. The extra purification step will help remove most (usually all) of the remaining contaminants, which might stabilise your target protein.
That said, some proteins are intrinsically unstable, in which case you might be better off trying to purify a homologue from a related organism. Also, large multidomain proteins are often unstable and this can be circumvented by expressing the individual domains rather than the full-length protein.
As Antonio mentioned include Protease inhibitor cocktail especially in lysis buffer and include SEC step after Affinity chromatography.
To this, I will add,, during ur IMAC give more stringent washes like 20 cv of 10mM, 10 cv of 20mM, 3-5 cv of 40mM and 3-5 cv of 70mM Imidazole washes before elting ur protein at >150mM Imidaole. Optimize ur washing conditions, so that u get maximum purity of ur protein of interest at Affinity level itself.
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