I am currently working on a protein which is overexpressing well in E. coli BL21 (DE3) RIL cells with 0.5 mM IPTG. It is cloned in a pET28a expression vector. But the protein is always coming in the pellet fraction during solubility analysis. The pI of the His- tag protein is 7.7. Things that I have tried till date and it didn't worked are-

- Culturing the cells (containing the recombinant plasmid carrying the gene of the protein) in different temperature like 16, 20, 25, 30 and 37 (degree celsius).

- Buffer types- Citrate (pH6), HEPES (pH7), Bicine (pH8.5) and Glycine (pH 9). These buffers have been used individually on cell pellets obtained after culturing at 16, 25 and 37 (degree celsius).

- 8M urea in HEPES (pH6.9) lysis buffer on cell pellets obtained after culturing at 25 (degree celsius).

Other important point- Cell lysis method- sonication.