My protein is forming soluble aggregates, i have tried upto 300mM NaCl, 2mM of beta-mercaptoethanol, or DTT, with 10% glycerol, 35 mM Tris-cl (pH-7.4), and 1mM PMSF. PI of my protein is 5.6 . My protein has a flexible N-terminal alpha-helix. I am using E.coli BL21 DA3 strain for expression. i am using 0.25mM IPTG for induction. Please let me know if you have any suggestion. it would be a great help
Thanks in advance
Deepak
I'm confused by your wording "soluble aggregates" are contradictory, proteins are typically soluble OR aggregated. Do you mean an INsoluble aggregate? This is much more common with strong promoters in E. coli. As such there are several good reviews of methods which MAY create a soluble version of your protein in E. coli. See here Chapter Overcoming the Solubility Problem in E. coli: Available Appr...
and related article tab. I've had excellent luck using Studier Autoinduction media for pET systems, usually at low temperatures, good luck!
Hi, I agree with Edward, I think you mean insoluble ( inclusion bodies), which is a common issue for recombinant proteins. First, try to use a lower concentration of IPTG and incubate your bacteria at a lower temperature, such as 15 C; this could help if not, you may have to change the expression vector or expression cells. Another strategy by forming a fusion protein ( link your interested protein with a highly soluble protein). Also, you can use a signal peptide to express your protein in periplasmic space.
@edward michelini @Husam Sabah auhim
Hi,
I meant protein is not going to the pellet fraction. It is soluble very nicely, on SDS-PAGE i can see the desired band. But when I do a size exclusion chromatography protein goes to void volume. In NATIVE PAGE also it is stuck in the well. They are called soluble aggregates because these are soluble in nature, but in solution these proteins are not in monomeric form.
Dear Deepak Nayak ,
You are absolutely right “soluble aggregates” is an official term. According to for example https://pef.facility.uq.edu.au/research-services/protein-purification/protein-aggregation it is described as “Aggregates can be categorised as either “insoluble” (able to be removed by centrifugation or filtration) or “soluble” (not easily separated from native protein).”
For a comprehensive overview of basically everything of what you can do to prevent/solve aggregation issues:
Mario Lebendiker, Tsafi Danieli (2014) Production of prone-to-aggregate proteins, FEBS Letters, Volume 588, Issue 2, Pages 236-246, https://doi.org/10.1016/j.febslet.2013.10.044
https://www.sciencedirect.com/science/article/pii/S0014579313008144
The quickest solution might be found in Table 3.
Best regards.
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