You should try first all the points in the previous answer. If nothing works and you still get inclusion bodies, remove urea slowly using dialysis. Dialysis buffer is not unique and largely depends on your proteins. On-column refolding is also an option if the proteins are his-tagged.

Dear Amit

as Aliakbar told you, to help you, we need to know more details about your protein name, the sequence that you cloned in the vector.

e.g

- Are you expressing the full lenght protein? A domain?

- Is it you protein a membrane protein or it contain membrane regions?

- Is it your protein rich of cysteines?

- Is it you protein a enzyme with an activity that could be toxic for E.coli or it have to bind some co-factor for its stability and activity?

. Which expression and the lysis conditions are you using?

There are a lot of different reason because a protein can go in the inclusion bodies and if it is true that refolding from urea sometimes work, many times is important take in consideration the previuos answers to desing domain and expression protocol.

Good luck

Manuele

If you are over-expressing, to maximize protein yield, then inclusion bodies are how E.coli stores excess protein that is foreign and that it has enough of. You will then need to unfold and refold in chaotropic salt like 6M guanidine HCl or 8M urea.

I agree with Mr. Yazdi in this regard.

I think ine continuous flow process is a fantasy since you would need to lyse the cell and then unfold and your cellular debris would eventually clog everything up.

Best strategy is successive unfolding and then refolding dialysis steps slowly over several hours. If you try to rush it into continuous lateral flow, you are going to sacrifice your yield dramatically.

That will happen no matter what strategy you use but you could try other suggestions of lowering incubator temperature during expression or cutting back on IPTG inducer concentration if you are determined. I don’t think continuous lateral flow is a good idea, though.

You can also try a vector with periplasmic sequence (pelB) but for limited quantity in soluble form.

Dear All

Thank you very much for helping and your precious suggestions. I am expressed Mtb protein in pQE30 vector. I already got few protein in cytosolic form and now trying with suggested protocol to get other also.

Thanks & Best Regards

Amit

Amit,

I feel strongly that my colleagues on this thread have given you outstanding advice that will help you to achieve success. If you still are uncertain as to specifics, another option is to check Current Protocols in Protein Science and the Maniatis manuals for Molecular Biology. The online versions I believe are updated with the most recent research but might involve a fee for access.

Best of Luck,

Don

Donald Bush

Thank you very much

best

Amit

Dear Amit,

could you share what factor did you changed, that helped you to get a soluble protein? I believe that others may be interested and find it useful.

Thanks in advance!

Dear Jan Binovsky

Once I optimize the protocol, Sure I will share it here.

Thanks for your suggessation.

Best

Amit

I used a trick that worked for me. Well you can aim at a slightly higher ratio in soluble fraction. What I did was, I performed a late induction protocol. I let my culture grow unto 1.0 OD which is late log phase without any induction then let cool the culture to 25 degrees in a slow shaking. Then I induced with 1.2 mm IPTG overdose. Harvested in half an hour got a good yield in soluble fraction. That much high IPTG is toxic but at 25 degrees cell die slowly and produce enough protein and due to high culture density I could get a fair yield of soluble protein. My protein was not active when refolded so had to try a lot of things. Best

Dear Sir

the above is an excellent discussion thread !!

I cannot really add anything to the insight above other than to say

• When I expressed protein in E coli by IPTG induction, growing for 20 hours at 18C-21C minimised the amounyt f insoluble inclusion fraction
• Include a full SOP that I used to use and incorporates much of the above and other background materials