Optimize the OD, Check for codon usage first

One of the reasons that once happened to me was contamination.

By the way, how long did it take you to reach 0.3 OD600?

Hi Rhudith Cabulong

                Check the following things for your research,

1. Check for Codon optimization,

2. Cloning or expression vector chosen,

3. Antibiotic concentration recommended for your plasmid

4. Post induction, keep the culture in different temperature, their are studies reported for better induction at lower temperatures.

5. Try to do plasmid isolation from your clone and do restriction analysis for your gene of interest,

6. Try isolating whole cell lysate, periplasmic, cytoplasmic samples and run PAGE to know localization of your protein

7. Did you include signalling peptides which direct the protein to secret out. like SEC Y, PelB, etc

Hope this will be helpful

All the best

1) May be your protein cells are lysed due to some contamination or detergent. 

2) Change your loading dye.

Is the protein soluble? Sometimes recombinant proteins form aggregates or even are targeted to inclussion bodies that are really difficult to extract. Another possibility is that the IPTG induction is not working, try with another batch of IPTG or raise its final concentration to 1-1.5 mM

You could also try lysing your culture directly in SDS-PAGE buffer (yes, the blue one), and if you've got an antibody try detecting your protein by western blot.

Hope this helps

Estanis

Dear Rhudith,

How can you tell on the SDS-PAGE that your protein wasn't expressed?

Yes my protein is soluble because I have other clones that has the same gene but in different plasmid under the Tac promoter. In that clone, the protein was expressed everytime 0.5mM IPTG is added and I can visibly observe the protein using SDS-PAGE. However, when I tried to clone the same gene to another plasmid, which is this pACYCduet-1, my gene of interest is nowhere in my SDS-PAGE

It took 3 hours for OD600 to reach 0.3.

The gene that I am using is already codon optimized and during my sequence alignment, I used the codon optimized sequence as reference and my sequence results aligned to it.

However, I wasn't able to include a tag for this protein because I included its stop codon.

pACYC is a low copy number vector.  Maybe that alone causes your yield to fall below the detection limit? 

I would suggest do a western blot to be sure that protein is not expressed at all. If it shows protein, try inducing for longer duration and after OD 0.6 in superbroth or TB. You can try increasing IPTG also.

Thank you everyone. I will try your suggestions.

I would perform the induction at a lower temperature. Sometimes if there is too much of the protein being expressed, as might be the case with the different vector you are using, the protein will be put in inclusion bodies. The protein won't be soluble and you may have to extract the protein from the insoluble fraction and reconstitution it (which is a pain). Try the lower temperature for a longer period of time, this helped me. 

Hey Rhudith!

Since you use a duet vector, are you trying to co-express another protein? Can you be sure that it is not that protein which causes your troubles? I guess you have a cloning intermediate with only the one or the other protein sequence. Did you already try to express them individually from the pACYCduet-1 plasmid?

And did you consider whether your expression strain is still appropriate for pACYCduet-1? Just in case you did not yet check for this.

Besides, I agree with the earlier recommendations. Especially, since you are facing troubles with overexpression, try western blot analysis for expression condition optimisation, if you have an antibody raised against his- or s-tag (both provided on the plasmids) available.

Best regards,

Christian

Thank you everyone for your suggestions! I already found out the cause of the problem. Yes Christian, it is my expression host that has a problem. lambda DE3 gene was not integrated into the chromosome of my host.

Thank you everyone for the suggestions.