See if you get some protein in the soluble fraction by Western Blot or by loading a large volume on His-Trap affinity chromatography. Maybe you have a low yield.

However, it will be easier to work on the soluble fraction than in inclusion bodies.

Nicolas Poirier Thank you for your suggestion. Is there any way to increase the yield during expression?

Inducing overnight at low temperatures can help with protein folding. You can screen your conditions by WB and then optimize your yield by choosing a Terrific broth or other medium. If you have a bioreactor you can use a feed batch strategy strategy to improve your protein expression.

Your protein is from a thermophilic organism. those tend to have quite strong hydrophobic cores among many other stabilising elements.

it is thus possible that your protein misfolds halfway through the expression.

A few tricks you could try :

- adding 2-3% (v/v) of EtOH in your culture ~1h before induction. that shock can do wonders with stable protein production (such as causing the expression of native chaperones to a higher extent)

- cold shock before induction (10-15 minutes on ice)

- adding 1% glucose to the medium

- co-expression of chaperones (the Gro series for instance, there are plasmids that can help for that, such as in https://www.takarabio.com/products/protein-research/expression-vectors-and-systems/protein-folding-kits/chaperone-plasmid-set, see attachment)

- If you see many cysteines in your sequence, try Shuffle or Origami strains (though they grow slower).

Hello Prayatna, more information would be helpful. Can you kindly share an SDS-PAGE showing the uninduced and induced cells? Are you transforming BL21 fresh and checking for expression of your protein from multiple colonies? If a protein is toxic, cells with mutated T7 RNAP will be selected so avoid the stress. Try adding 100 mM glucose to your overnight cultures and avoid subculturing too much. Good luck!

Prayatna, just to follow up, when you say the sequence was confirmed, did you also confirm that the T7 promoter and terminator are intact? I also assume the BL21 cells are bought commercially? If they are lab-made cells the T7 RNAP may have become mutated over time. Please provide anymore information that you think may be pertinent. Thanks!