Hi Aparna Gunda I wonder if you try IPTG induction system, therefore you should use IPTG to induce the expression of recombinant protein or you may try different temperature to see the induction of recombinant protein.

Dear Aparna

to provide to you reliable suggestion we need to know some more details about your protein sequence and the expression protocol.

When you mean IPTG induction system, do you mean vector carrying a T7 promoter and expression performed in BL21(DE3) cells?

Which is the MW of your protein?

It is contain transmembrane regions or signal peptides? and in case, did you remove the signal peptide in the E.coli expression clone?

Did you check the sequence for rare codon?

Did your protein contain many cisteines?

When you wrote, even at 18°C, mean that you tried several induction temperatures? Which?

i ask you this question just because details some time make the difference for IPTG induction with T7 promoter you need a specific strain that is able to express the T7 polimerase (eg DE3 strains) while other IPTG inducibile promoters as the TAC are working in all e.coli strains and therefore also in normal BL21.

Considering that your protein is eucariotic is possible that the DNA sequence contain a lot of rare codon that can make difficult the expression in E.coli and in this case you have to:

- try the Rosetta strain

- Buy a codon optimized synthetic DNA fragment.

If did you already check and optimize this step, one other possible solution to improve the protein expression is use a N-terminal fusion tag as GST, Trx, GB1 and insert a TEV recognition site to remove it after purification.

Using this approach in the past i was able to successfully express several human protein in E.coli

if you are interested to see an overview of different tools (including TAG) that you can use in E.coli, you can see the following video

https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG

while if you would like to predict rare codon or trasmembrane regions you can find some usefull softwares in

https://www.blogger.com/video.g?token=AD6v5dyPGWsbPCAGPM4bhC7qrFmUL9TIfeArxzSqZ-ao_K6iC3z597YtqkSB2QIvBIxy9TVU52O9By7lhSuO-TFmnmd1-xRgRxnhW6uEon8lV-Lk-zMV2mZq1MT0o4gwaT38-wIRIIY

both video are available on my blog: ProteoCool

ProteoCool: Video Playlist

good luck

Manuele

thank you Manuele.

Earlier I tried 37 degrees with glucose in the medium. Codon optimisation is done

using the right bacterial strain