Did you only try SDS PAGE on the soluble fraction or did you use both soluble and insoluble fractions? Also if you haven't I would do a western blot, since SDS PAGE alone can't really tell you if expression is absent vs. poor.

Check if start codon is present in the sequence.

Also, lysing the cells and running the pellet fraction in SDS PAGE might help in case protein expression is low and it is forming inclusion bodies. If there is tag in the protein, running the lysate through an affinity column might increase protein concentration, thus making it easier to view in gel, if it is soluble and being expressed at a low rate.

Dear Pradeep

there are many possible reason because a protein do not express in E.coli and the solution is not obvoius and not ever the same

to provide a reilable answer we need to know more details about your protein and the protein domain that you cloned.

Is it a membrane protein?

Did you cloned the full lenght protein or a protein domain?

Did you had any C- or N- terminal tag?

Is it contain cisteines involved in S-S bonds?

best regards

Manuele

Thank you Alexandra Johnson for your suggestion. I will do a western blot to check the expression.

Manuele Martinelli yes it is a membrane protein. I am cloning full-length gene. His tag is present at both N and C terminals. one cysteine is present in my gene.

Bidisha Chakraborty Thank you for your suggestion but I have tried this and still unable to get an expression.

Dear Pradeep

membrane protein are an hard target!

Did you observe any stop in the E.coli growth after induction with IPTG?

The use of a N-term fusion tag as MBP, may help to improve the expression level. Some proteases, as TEV are able to work also in presence of detergents (see attached paper).

In my opinion, it depends a lot from your protein properties:

If your protein is a transmembrane protein with many trasmembrane domains (eg GPCR) probably cell-free expression (give a lock to the papers of Prof. Frank Bernhard - University of Frankfurt ) may have high probability to succeed.

If your protein contain a single or few transmembrane h-elix domains, you can try to clone and produce the soluble domain.

good luck

Manuele

From the given information, I wonder if your BL21 comp. cells are old and not working! Also, consider sonicating the bacterial pellet in mild detergent , say 0.1% NP-40/PBS, and recheck your transformation. You can use some positive control and negative control. Hope it helps. Good luck.