Are you sure the plasmid is good and the insert is in-frame? Have you sequenced it?

If it is good then you just got to scale up your protein expression to 6 - 9 liters of culture and pull the protein out of everything else with an affinity tag like a polyhistidine tag using Ni-NTA resin and an imidazole elution. Sometimes you got to give up optimizing your protein expression on a small scale. Some proteins just don't express well and never will. They will never be as forgiving as lysozyme or green fluorescent protein.

Dear Adron Ung,

Thank you very much for your response. I will see if this works.

You don't mention how you are looking for the protein to determine it is not present. It might be the protein is aggregating in inclusion bodies or is membrane inserted, in which case it would not be found in the soluble fraction after lysis. So be sure you aren't discarding it.

Also sometimes you have a transformant that is not expressing. Retransforming the plasmid into fresh cells and screening fresh transformants can sometimes help.

Adron Ung

I had sequenced and found the insert to be there.

That is not what I asked. I know the insert is there, but how are you determining whether the protein is being expressed or not?

Michael J. Benedik.Hello Sir, I replied to Adron Ung.

My supervisor told me first check the whole cell lysate i.e once after induction, the cells (5 mL) are harvested by centrifugation followed by PBS Wash and resuspending of the pellet in 200-300 microliter of PBS.

After resuspending, I took an appropriate volume of these cells plus protein loading dye and boiled it for 10 minutes.

After the boiling step, I centrifuged the sample at 10,000 RPM and 10 Minutes. Analysed this sample (15-20 microliter) in 12 % SDS-PAGE as it is 72 kDa protein.

The whole cell lysate is the proper way to assess, so that is ok. I would suggest you also retransform and test 5-10 independent colonies to see if any express.

Dear Ganesh Kumar

can you provide as some more information about the protein domain that you would like to express?

for example:

- MW

- Does it contain many cisteines?

- Did you cloned the fulll lenght protein or just one domain?

- Is it a membrane protein or soluble protein?

- How many rare codons are present in your DNA sequence?

Because each protein is different and a possible solution (eg addition of TEV cleaveable a N-termnal fusion tag as GST, MBP, GB1 or Trx) which may improve expressione level) can succeed if your protein is soluble and do not contain many S-S bonds while will fail in the opposite case, since E.coli cytoplasm is too reducin g to form S-S bonds and in this case you need to try periplasmic expression of more to an eucariotic host.

Ciao

best regards

Manuele