This question gets posed repeatedly at RG, and there is no magic bullet to addressing this problem. You have to tackle it empirically with some guidance based on the known properties of your protein.

I'd suggest you look at other RG threads on this topic, including https://www.researchgate.net/post/How_can_I_get_a_eukaryotic_protein_in_soluble_form_which_was_expressed_in_Ecoli

The most popular strategies are 1) try to use a globular domain such as MBP as a tag to improve solubility 2) try induction at lower temperatures 3) express only the domain of interest of your protein to remove unstructured or highly aggregation-prone regions or 4) perform an inclusion body purification followed by a refolding protocol.

In my experience, the last option has the highest success rate, but there are cases where you can tweak the conditions to coax the recombinant protein into a soluble phase.

This question gets posed repeatedly at RG, and there is no magic bullet to addressing this problem. You have to tackle it empirically with some guidance based on the known properties of your protein.

I'd suggest you look at other RG threads on this topic, including https://www.researchgate.net/post/How_can_I_get_a_eukaryotic_protein_in_soluble_form_which_was_expressed_in_Ecoli

The most popular strategies are 1) try to use a globular domain such as MBP as a tag to improve solubility 2) try induction at lower temperatures 3) express only the domain of interest of your protein to remove unstructured or highly aggregation-prone regions or 4) perform an inclusion body purification followed by a refolding protocol.

In my experience, the last option has the highest success rate, but there are cases where you can tweak the conditions to coax the recombinant protein into a soluble phase.

Sonication will not result in complete lysis of cells. Therefore protein will be observed in pellet obtained after cell lysis. What is the % expression of your protein in total cell protein.

You should look first into the intrinsic properties of your protein. Is it glycosylated? does it have disulphide bond(s)? is it hydrophobic in nature? when you clone it, does it have unusual/rare codons? etc etc. Based on those considerations, you can devise what plasmid to use, what protein tag to recruit (if you want one), use different E. coli strain (Rosetta, Gami, or combination). Like Daniel said, it's rather trivial.

When you found the protein in the pellet after sonication, did you check whether it is insoluble or somehow bound/anchored to the cell membrane? You can double your sonication step; first just in standard buffer (PBS, TBS, alike), after that you can recover the pellet and sonicate again in the presence of low concentration of detergent (0.1% triton X100 or alike). Even when I pursued refolding step, I do these steps as it will provide me better starting material for refolding.

In my previous work, I was told to include 10% glycerol and some reducing agent (DTT, about 1-2 mM) in the buffer which I used for sonication. I personally don't like that idea as I may recover some proteins in the soluble fraction but I don't know whether they are truly the correct one or simply some micro aggregates co-solubilized during sonitation.

Thanks for all for valuable suggestions. The % expression of protein in total cell protein is 20. I am following the protocol which is already been standardized where only 50% of protein is going to pellet. But in my case I can see only 20% in soluble form. I don't know where I am going wrong. I am using 1% triton X100 in lysis buffer.

Sonication condition is 50% Duty, 20% power for 35sec with 30sec interval.

Is sonication condition has any effect on the protein precipitation?

You are getting 20 % protein in soluble form that means cell is making protein in soluble form. Now important is your method of analysis. Protein which you r saying is going in pellet may have both soluble form and Inclusion bodies. Therefore you need to find out % of cell lysed. If only few cell are lysed then you will get misleading results. For better lysis increase cell to buffer ratio or use other methods of cell disrutpion.

You are saying there is 20 % protein expression out of which 20 % is in soluble form . Then I dont see any problem.

I mean to say 20% of the total cell protein is our protein. however only 20% of total expressed protein is in soluble form.

Please let me know how to check % of cell lysis.

If you digest your bacteria with lysozyme prior to sonication, or switch to using a French press, you can greatly increase the efficiency of cell lysis. This might help, but I don't think will completely resolve your problem. Try to see if induction at 25 or 30C improves the fraction of soluble protein.

Also, make sure your samples are not overheating during sonication. Heat-induced denaturation can cause your protein to aggregate and become insoluble.

In additon to the suggestions above, you could try harvesting the bacteria at different times after adding IPTG. There could be a point where most of the expressed protein is soluble.

Switching the expression vector to one that coexpresses a chaperone (http://www.abvector.com/InclusionBodiesProblem.htm) can also help (though I would suggest trying Daniel's and Steingrimur's suggestions first).

Try inducing with IPTG at 22 -25C and harvest after different time intervals say after 4, 5. 6 , 7 hrs after induction. Plz determine final OD for all these samples.

Thanks Daniel I think problem lies in sonication. I reduced the duty% also increased the time intervals between 2 cycle. And also I checked in 2 different sonication condition where in one of the condition protein were going more into the pellet though the cell lysis was complete.

Thanks everyone for valuable suggestions.

Try various ratios of cell pellet and lysis buffer

Hi Manjula,

you could also try reducing the IPTG concentration as 0.5 mM is quite high. Going as far down as 0.1 mM IPTG might help. Some inclusion bodies form due to an extremely high expression rate, which you can prevent by using less inducer.

Best,

Karim