Hi,

I think troubleshooting in the case like this one is more or less standard: Confirm the sequence (exclude point mutations that lead to termination of ORF) of the cloned insert in the resulted pDNA used for transformation. If the sequence is correct, try and use BL21DE3 pLysS bacterial strain that allows for more stringent control of protein expression (less promoter leakage). That may be helpful if the protein expression is toxic to the cell (as DnaA activates initiation of DNA replication in prokaryotes, its over-expression might be toxic?). Next, use room temperature induction vs short time (1-3h) 37oC induction AFTER the cell culture is almost suturated (OD600~1.0-1.1). If your protein is tagged, use WB versus the tag to confirm the presence of the protein.

Good luck.

Toxicity can be a problem. I didn't try with bacterial replication proteins but overexpression of mammalian ones in mammalian cells is often toxic. In addition, did you also test your pellet in case your overexpressed protein is insoluble?

It May be a toxic protein. Almost never its good for the host cell to overexpress a itself regulatory protein. The easiest way to prove Whether the protein is toxic, is comparing the growth (duplication time or OD ) between a culture induced with IPTG and other non-induced (or better, repressed with glucose). If the protein is toxic, non-induced control should reach a higher OD or have a much lower duplication time.

Regards

Is there no over-expression when you run a SDS-PAGE gel? Did you check the sequence of the cloned gene? As Oleg mentions, using normal non-proof reading Taqs can introduce errors. Unfortunately, these will be the ones selected when transformed as they are less toxic than the correct forms. Good luck!