Protein doesn't express

[Marco Oliveira]

Please, I need help. I bought a synthetic gene from Genscript which came already cloned in a expression vector, pET28a. I'm experienced in protein overexpression, I'm following all standard methods for protein overexpression. Other proteins are been overxpressed normally at my lab, however this particular one isn't. I double checked the sequence, the codons are optimized and everything looks fine. However, the protein does not express at all in BL21(DE3) background. Any clue or suggestion?

Edited Wednesday at 08:38 PM by Marco Oliveira

[CharonY]

There is a possibility that the protein is harmful to the cells. Are the proteins membrane proteins and/or is aggregation likely? Is the vector present in cells when not induced? And does it remain if you induce?

[Marco Oliveira]

1 minute ago, CharonY said:

There is a possibility that the protein is harmful to the cells. Are the proteins membrane proteins and/or is aggregation likely? Is the vector present in cells when not induced? And does it remain if you induce?

Thank you CharonY for your response. I don't think that the protein is toxic, since other works have expressed homologues with no trouble. It isn't a membrane protein, and I'm not sure if it aggregates. It is a human protein. I think that the plasmid is stable as I add kanamicin during the induction. I may try to increase the antibiotics concentration a little bit.