Dear Jyoti Verma

if the theoretical DNA sequence is ok (first met and stop codon are in the same frame?) , probalby it depends from the properties of the protein sequence.

It contain many rare codons?

Is it a membrane or soluble proteins?

It is contain many cisteines?

best regards

Manuele

Dear Jyoti Verma,

Based on your description, it seems you have followed all the necessary steps for cloning and confirming the presence of your gene in the plasmid. However, the lack of protein expression after induction can be due to several factors:

1. Check for rare codons as suggested by Manuele Martinelli.

2. The protein you are trying to express might be toxic or forming inclusion bodies (insoluble aggregates). Did the SDS-PAGE show protein in the total cell lysate?

3. Even though you confirmed the insert by colony pcr and restriction digestion, it is best to sequence the entire insert by Sanger sequencing, to rule out mutations within the insert.

4. Was the induction done with 1mM IPTG, and for how long? Test lower IPTG concentrations (0.1-0.5mM), time of induction, and lower temperatures (16-18°C) in a small-scale expression experiment to optimize conditions for protein expression.

5. It could be possible that your protein is degraded. To prevent this, add protease inhibitors during lysis.

6. Lastly,If the above steps do not enhance your protein expression, you can also test auto-induction media using 10% lactose.

Hope it helps! Good luck!

Best regards,

Samiya.

Restriction digest and PCR won't tell you if you have any small changes in sequence (e.g. SNPs, small InDels, etc.).

Sequence your plasmid to make 100% sure the insert is in frame and in the correct orientation.

Choose several colonies (4-8) and sequence the plasmid in all of them. Once you find one that's what you want, continue with that one.

And check the IPTG, it can degrade quickly from repeated freeze-thaw cycles. When in doubt, make it fresh.

Good luck!