What was OD600 before harvesting? What was the final concentration of IPTG? Did you load samples from the total, soluble and insoluble fraction of the cell lysates on SDS-PAGE gel?

Did you verify cloning of your target gene by sequencing?

It seems difficult to suggest anything on the basis of given information. Could you please write the following details. 1. The percentage of overnight culture added as inoculum to the induction flask?...2. OD 600nm at the induction time?...3. Shaking rpm of induced culture?....4. The ratio of space between culture and air spce in the culture flask?...5. The induction procedure and the time given after the start of induction?.....all these parametere matter to smaller or greater extent.

Simple BL21 is not DE3 also cspA is from E. coli rather than phage origin. so, I assume the protein synthesis in case of cspA promoter is induced by lowering temperature, not by IPTG. If you will go near 20 celsius, you will shift away from the optimal growth conditions of BL21, slowing down the E. coli synthesis machinary. I dont know, whether you continue at low temperature? if yes, then you need an overnight induction time for better results.

Dear Suyash Patil

Please you can find the attach file. Hope it will be helpful

Dear Experts,

Thank you very much for the information. The details of the protocol I follow is:

I have sequenced the final vector for its accuracy and orientation. I am using BL21 DE3 cells specially recommended for protein production. I am using OD=0.4 while inducing the culture with IPTG (final conc. 1mM). I am using small volume of 3 ml culture in 10 ml tube to check whether the protein is induced or not. The induction protocol is 15 degree celsius overnight at 180 rpm. I have used SDS page gel to check this using un-induced sample as control.

I am using nanodrop to check the OD values.

Thanks,

Suyash

Dear Suyash Patil,

The strain BL21 (DE3) is compatible to T7 promoter based plasmids. The cspA is E. coli based promoter. I think there is a technical issue. We can induce protein with IPTG in T7 based promoters. I doubt, if we can use IPTG for cspA promoter to induce protein expression. I have gone through the sequence of your plasmid, there is cspA promoter, I could not find any sequence for T7 promoter which is essential for DE3 strains. As you know, T7 is basically a lysogenic addition of T7 phage RNA polymerase in the bacterial genome. Once we induce by IPTG, we start the production of T7 RNA polymerase (a hyperactive polymerase which is highly specific to T7 promoter). In your case, the RNA polymerase may be produced by induction, but having no T7 promoter, it fails to deal the transcription of your target gene. I think you may replace the strain DE3 with simple BL21 (non DE3) and induce at low temperature (below 20C) while using cspA promoter based plasmid or use pET series or any T7 based plasmid with DE3 strain. I really wish you good luck as I can feel the sufferings of having no expression......!!