Hi satya

Its quite possible that because of any reason your insert get mutated for a stop codon in pETa vector, try to resequence your expression clone. If your clone is fine, than you can try expressing your protein using other strains like BL21 pLys, Rosetta etc

Hello,

i agree with Roshan, try re-sequencing your construct. Mutations in the promoter of the construct may be one reason and definitely generation of a new stop codon within the sequence can also be one. The simplest way would be to re-clone in a different vector series. For mycobacterial proteins i always find pET23a a better option. 

Thank you Roshan and Suhail.

I agree with Roshan and Suhail, the first thing you can do is to re-sequencing your construct. If you are sure that your vector matches your expression strain and that there is no mutation in your sequence, but you still have no expression, you can try out different conditions and hope that something happens in one of them. The usual suspects are protein toxicity and codon bias. See more information form troubleshooting of bacterial protein production.

The problem of protein toxicity may arise when the bacterial protein performs an unnecessary and detrimental function in the host cell. Most expression problems are also the result of protein toxicity. With optimization of expression vectors and host cells, we estimate that 80% protein yield problems are the caused by protein toxicity.

The problem of codon bias is also a common reason of protein expression problem. By codon optimization, we can improve protein expression level in living organism by increasing translational efficiency of target.

http://www.biologicscorp.com/blog/troubleshooting-bacterial-protein-production/

http://www.biologicscorp.com/codon-optimization-technical-platform/

Thank you Nancy for sharing such an important link. Cheers!!