Co-expressing the protein with chaperones (GroEL/ES) can be helpful. Also, reducing the temperature during expression sometimes helps.

Thanks a lot.

Do you have a strain that overexpresses GroEL and GroES?

You can also try to use the Lemo21 strains, they carry a plasmid which allows you to fine tune the expression levels of your protein of interest. For expression at very low temperatures, there are the ArcticExpress strains available. If the inclusion body formation is due to insufficient disulfide bond formation, you can try periplasmic expression using the ompA signaling sequence or you can try the Origami2(DE3) strains. 

Hi Mr. Mordechai,

Kindly follow this link for the detail description about the E.coli BL21 strain and its functions.

http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html

You can also try these methods to increase the solubility level of your protein:

(1). You can use 10%-20% of glycerol in your media. Viscosity provided by glycerol lowers the growth and helps in solubility.

(2). Try to induce your recombinant protein with low concentration of IPTG such as 0.1-0.5 mM IPTG.

(3). I have faced similar situation several times there i have changed growth media, sometimes it works. try to optimize the solubility in different media such as LB, 2XYT, Terrific Growth, S9 etc. You need to optimize the different concentration of IPTG for different media.

(4). One of my protein i got soluble in AIM (Auto induction Media). Its quite good for high yield soluble protein. There are different AIM media available such as AIM-LB, AIM,2XYT, AIM-TB. Try these, i am sure you will get good response.

(5). Lower down the temp to 16-23 degree after induction, Sometimes lowering the temp works better.

(6). You can use 1-3% of ethanol, In some cases ethanol is reported to decrease the inclusion bodies formation. Add 1-3% of ethanol while inoculating the culture. Growth will be less which ultimately helps in solubility.

(7).In some cases less amount or less concentration of osmolytes such as glucose, sorbitol, mannitol etc can helps in solubilty. Start with less concentration of these osmolytes.

(8). You can also try the heat shock method to improve your solubility. Grow your culture till 0.2 OD in 37 degree then grow it at 42 degree till 0.6 OD then divide it into 3 fraction induced with 0.5 mM IPTG and incubate at 23 degree and 37 degree 42 degree for overnight, 5 hrs and 3 hrs respectively.

(9). Try different expression host according to your promoter for T-5 use M-15, SG13009, XL-GOLD, XL-BLUE, TG-1 for T-7 use BL-21, BL21(DE3), ROSETTA, CODON PLUS etc. there are various cells. optimization depends upon the availability of these cells at your lab.

(10). Optimize all condition if you won't get any positive response then ultimately you need to change the Tag or vector.

I hope anyone of above methods should click for your work.

For any further queries you are welcome.

Good Luck.

Hi Mordechai,

You can try BL21-AI, which offers tighter regulation of protein expression than most other strains, or Lemo21, as someone has already suggested. If you suspect that your inclusion bodies are due to improperly formed disulfides, you can try Shuffle or Origami. If your yeast protein is not codon optimized, then you can try Codon plus or Rosetta (or even Rosetta gami, which combines the properties of Rosetta + Origami). Co-expressing with chaperones might also help - these plasmids are commercially available so you can introduce them into any compatible E. coli strain. 

What usually works for me though is playing around with induction time and temperature as well as inducer concentration, E. coli strain, type of media and tag. From my most recent experience, after having tried all that, what ended up working was switching to autoinduction media and changing the tag to MBP. But each protein is unique, so you just have to try... 

Good luck!

A lot of good suggestions already made above but I would suggest to start with point no. 10 of the list above because you don't want to repeat all the screening steps for strains, media, buffer etc. for one construct after the other. Try multiple constucts (4-6 different fusion tags) first and see if there's any trend or if it's always insoluble. Screening of different conditions can be done at small scale easily in parallel using 24 deep-well plates.

If nothing works use another expression system, so while you're re-cloning your insert in E.coli vectors, you could (should) also generate some vectors for insect, yeast or mammalian expression (if one or more of these are available).

You are getting great suggestions. I'll just add that you could try using SoluBL21 cells. I've had a lot of success with this particular strain. Also, adding sucrose (0.2-0.6M) to your media will help to slow growth, which in turn, may produce a soluble protein fraction.

Using 0.5% glucose in media may help but then you must grow culture after induction for less than 12 hrs and more than 10 hours. In this time window you may get good solubility but after 12 hours of growth cells may lyse owing to acidic conditions of media due to glucose. Additionally you may keep your culture at 4 degree celsius for 20 mins before induction to induce chaperones which will help prevent inclusion body formation.

I guess that all possible strategies are already provided in the previous answers, so I would just like to add a review where the factors supporting soluble protein expression are covered:

Hans Peter Sørensen and Kim Kusk Mortensen, Microbial Cell Factories 2005, 4, 1

"Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli"

doi:10.1186/1475-2859-4-1

http://www.microbialcellfactories.com/content/4/1/1