Hello, I am trying to purify a 6X-His tagged protein using Ni-NTA chromatography. However, I obtain very less yield & concentration; and considerable protein in load flow through. Any suggestions to improve the yield and protein concentration in eluate fractions?
Below are the conditions used:
1. Protein contains pelB secretory signal, so gets transported to outside of the cell. Expression is induced using 1.0mM ITPG for about 5-6 hours at 37 degree C.
2. Supernatant treated with 8M urea for 3 hours before loading to Ni-NTA column.
3. Buffers used:
Equilibration - 50mM Sodium phosphate 300mM NaCl, 8M Urea pH 7.6
Sample load - pH adjusted to approx 7.6
Wash 1 - 50mM Sodium phosphate 300mM NaCl pH 7.6
Wash 2 - 50mM Sodium phosphate 300mM NaCl, 2mM Imidazole pH 7.6
Elution - 50mM Sodium phosphate 300mM NaCl, 100mM Imidazole pH 7.6
Appreciate any help!
Hi,
Is the expression optimised, have other conditions (media and temperatures) and concentrations of IPTG been trialled (0.1, 0.5 as well as 1,.0 mM)?
I would adjust the imidazole concentrations to reduce non-specific bining.
10 mM in sample buffer
20 mM in your wash buffer
and use 250mM in your elution buffer.
Culture conditions are yet to be optimised. I noticed some protein got eluted at even 5mM Imidazole in wash buffer.
Even found some protein in sample load flow through fraction, why is it so?
So, the concern is - why all the protein molecules are not binding to the resin?
Considering that protein expression as inclusion bodies is good enough, you are missing steps or doing things wrong. Inclusion bodies must be washed to removed contaminant proteins and cellular components. I suppose you are filtering or centrifuging the protein solution before feeding it to the column. Once 8 M urea is added, you are denaturing all proteins available, not just the recombinant protein. The thing is that these native proteins might have affinity for the metal ions and will compete with the recombinant protein (especially if the contaminants proteins are too concentrated). What is the size of your column? What flow rate are you using for sample application and washing? Even if the protein adsorbs, you are precipitating it once you wash without adding urea.
Dear Saaraj Gupta
why did you treated with the urea the supernatant?
in this way you induce protein unfolding and the protein can aggregate during the washing and elution steps?
did you tried to buffer excnahge the supernatant in a buffer similar your wash 1 but with out urea?
best
Manuele
If you are using pelB, the majority of the protein will be in the periplasmic space and can be purified by osmotic shock (go look at my nanobody papers). I don't know why you are including Urea, this step doesn't make sense. You should be washing your Ni-NTA column with 10-20mM imidazole, not 2mM. If you detect protein in the initial flowthrough, just reapply the FT a second time. I usually see 20% of input in my FT and after a second application it is almost 100% of input bound. Track your input and look at your wash steps for where it is going and you should be able to improve your yield.
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