There is no recipe to fix this situation. You can try (with the same cells):

Lowering the induction temperature

Lowering the inducer concentration

Dear Usha Kantiwal

the most simple solutions to improve the overal protein solubility in E.coli are

- lowering induction temperature (eg. 17°C or 20°C 24h)

- use a promoter able to modulate the expression rate of the protein (eg ara indubible promoter)

- use strains able to perform some post traslational modifications (e.g origami)

- fuse the protein domain with carriers with high solubility (eg. MBP)

- express the protein in the periplamic space by adding a leader peptide (eg pelB or ompA)

however the expression of soluble heterologous eukaryotic proteins in bacterial may fail due to several reasons and there is no a universal recipe to solve it and in some cases is preferable move directly on eucariotic expression sistems (eg Expi293 or ExpiCHO)

It depends a lot from the properties of the specific proteins, from the domain that is expressed.

Just some questions:

- Do you protein contains a lot of cysteines?

- Which is the MW of the proteins?

- Do your protein contain a leader peptide for secretion in eukariotes? If yes, did you removed it during the cloning.

best

Manuele

Dear Usha Kantiwal

Optimizing the expression system to avoid the solubility problem is always a good idea. However, whatever system you end up with, a protein with solubility issues will always be present in the sample in two states : aggregated and monodisperse (it could be a monomer, a dimer, ...). Therefore, another option to explore is to try to separate the two states, after overexpression, during the purification process.

The use of columns may be difficult because of the risk of clogging, especially in the gel filtration. However, ultracentrifugation (>200.000 x g for at least 10 to 15 hours) has been shown to be efficient in separating aggregated and non-aggregated states of an overexpressed protein.

Best

Yves

What you want is to increase the soluble fraction of your protein and reduce the formation of inclusion bodies during expression. The fundamental principle here is to slow down the protein expression.

1. Lower Expression Temperature – Reduce the temperature (e.g., 16–20°C)

2. Optimize Inducer Concentration – Lower the concentration of IPTG or arabinose to regulate expression levels.

3. Choose Low-Copy Vectors & Strains – Use vectors with a lower copy number and E. coli strains optimized for soluble expression (e.g., BL21(DE3)pLysS, ArcticExpress, or Rosetta for rare codon optimization).

4. Co-Expression with Chaperones – Co-express molecular chaperones (e.g., GroEL/GroES, DnaK/DnaJ/GrpE) to assist in proper folding.

5. Use Alternative Promoters – Choose weaker promoters (e.g., lac, araBAD) instead of strong T7-driven expression to slow down protein synthesis.

5. Consider fusion tags (e.g., MBP, GST, SUMO) to improve solubility.

By implementing these strategies, you can significantly improve the yield of soluble protein while minimizing inclusion bodies. Let me know if you need more specific recommendations!