Hey,

I would like to express and purify the protein that is encoded on nuclear DNA and then transfer to the mitochondrion.

The full length CDNA starts with the transit peptide. I assume that if I leave this peptide at the beginning of my ORF and perform overexpression in mammalian cells, the protein will be transported to the mitochondrion, which may result in cell death due to protein overload. Even if the cells survive expression, I have no idea how to clean the mitochindrium protein.

The only idea I have right now is to change the transit peptide into a signal peptide from some other secretory protein, and I hope that this will lead to protein secretion.

An additional difficult feature of this protein is that it must form trimers to be enzymatically active. If the signal peptide is not truncated during protein secretion, the trimers may not be formed.

Does anyone have experience in this case and will be happy to advise me?