is your gene encoded on the mitochondrial genome? or does it have any modifications as far as you know?

Hanna Alalam It is nuclear encoded and imported to the mitochondria. Any modification is not known but it has farnesylation motif in its sequence, which help in protein-protein interaction. But that shouldn't be problem.

ok try to use a weaker promoter like the pBAD system for protein expression. Also check the codon index of your gene (https://www.biologicscorp.com/tools/CAICalculator/#.XTwC7PIzZaQ), I know you used a rosetta strain but measure the index anyways. Use a low iptg concentration and grow the cells overnight (this is just to test toxicity). If all else fails you might need to express in a eukaryotic system. In this case switch to pichia pastoris and swap the n-terminal signal to a secretion signal (you can also remove it in ecoli if you think that might cause a problem).

What about expressing the his-taged version in yeast?

Remove the N-terminal signal sequence could help your protein expression, mainly for two aspects:

1. Mitochondrial presequences are removed in the matrix by the matrix processing peptidase. So, if your protein is targeting to mitocondrial matrix, its mature form will lack the signal sequence;

2. Proteins destined to the mitochondrial matrix are synthesized with amino terminal presequences that are amphipathic helices. So, it will not be functional in E. coli and the pre-sequence can potentially disturb the heterologous expression.

Best,

Ana

Sebastian Schmitt

Hi Schmitt,

I mean by "does not express" is, I could not evidently see the corresponding protein band on SDS gel. I tried pGEX system as well. I've to mention that its cytosolic homolog expresses well even with pET28 system.

Thanks,

Amit

Ana Paula Ulian Araújo

Hi Ana,

Thanks for your answer.

I was also wondering if I remove the presequence tag, but was doubting how does it make difference in bacterial system. I got confident with your system. I believe, although E.coli lacks the cellular organelles, the presequence signal would still able to create some hassle.

Best,

Amit

Hi Peter Kysel ,

We are thinking on the same line. I cloned this protein in yeast expression vector and, successfully complemented the homlogous deletion in yeast. But the question remains same, if I wanted to bulk purify it.

Thanks.

Amit

I have no experience with optimalization of expression in yeast. But from what I heard and read I can recommend puting the extra tags (His + FLAG/V5) on C-terminus.

Dear Amit,

I would modestly suggest you to make use of the Pichia pastoris expression host. It gives you a high expression yield. The pPIC vector has a signal sequence by itself; So you may not have to be concerned about the signal sequence in your gene (It may or may not function as expected). Additonally, His Tag purification could be possible as well. I faced a similar issue when I attempted expressing a protein from a filamentous fungi in E. coli; Later, Pichia pastoris expression helped me solve the issue of expression.