I'm working on isolating recombinant protein in E.coli (Ni-NTA)
I'am trying to purify recombinant protein that I overexpressed E.coli BL21(DE3)pLysS using the pCold1 vector.
The proteins have 6xhis on their N-terminus
The experimental conditions are as follows:
- E.coli strain : BL21(DE3)pLysS, vector : pCold1
- Grown in 500mL LB medium up to OD of 0.5
- Induction by 1mM IPTG at 37℃ for 6 hour
- Binding buffer : 50mM Tris, 200mM NaCl, 5% glycerol, 25mM imidazole, 1mM DTT, pH8.0
I elute with elution buffer using linear gradient ( 50 to 500mM imidazole)
Lane 1 : 225mM imidazole, Lane 2 : 250mM imidazole ...... Lane 9 : 425mM imidazole
But, even when the imidazole concentration is very high (over 300mM,), all the protein does not elute in the column.
it looks like histidine-tag aggregates.
Has anyone had any similar experiences ? I would appreciate any advice you may have. I'm so confused!!
I think you are getting good results. Most of the protein eluted in the earlier fractions at reasonable imidazole concentrations. That should be sufficient. The protein that came off later is a minor trailing portion.
When you use the pCold vector the induction conditions for protein expression should be done at lower temperatues (~15ºC) maybe this can increase your yield.
Thank you for the reply.
Hello Sebastian Schmitt
, Diogo Figueiredo , Adam B ShapiroProteins eluted with 200-275mM imidazole are in native form
Proteins eluted with more than 300mM imidazole appear as soluble aggregates.
After growing the cells to OD 0.5, I attempted expression at lower temperatures (15°C), but the Ni-NTA purification results were still the same.
------------------------------------------------------------------------------
I read a paper that proteins can aggregate in buffers containing high concentrations of imidazole.
For that reason, I removed the histidine-tag from the construct and eluted the protein with a low concentration of imidazole. and GPC was performed for 2nd purification.
As a result, the purity of recombinant protein was very high, and the previously observed soluble aggregates form did not appear.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies