Hi!! Im working with hemoglobins, is not exactly the same, but it´s also a hemeprotein. I don´t know what protocol are you using for you protein expression but, in my experience you can try adding Fe-Citrate during the expression. In case it doesn´t work, you can try adding hemin and reconstituing your proteins.

Good luck!

Delta-ALA is a heme precursor, added to express heme containing proteins.

In addition, addition of trace element mix containing FeCl helps.

And use lower temps, like 24-28 during induction.

For measuring heme there are many colometric assays, using hematin as standard. If using hemin, dissolve in 0.05N NaOH

https://www.sciencedirect.com/science/article/abs/pii/S0003269798929979

Irene Villar Thankyou for your suggestion. I am trying to add hemin to the media. Any idea how can I prepare hemin and adding directly to the media would have any effect on E.coli?

Alka Senwal When I was producing my proteins, I used to add hemin during the purification protocol. I mean, once I had the soluble fraction of the E.coli culture with my protein, I added 1 ml of 0,1g hemin /1 ml 0,05 M NaOH to the preparation. While you are adding it, you should have your proteins cold and stirring continuously. Then, I dialyzed the preparation in 50 mM Glycine pH9,5.

And that´s all.

Hope you find it useful.

Irene Villar Thanks for your answer. But I am wondering if this method also applies to protein containing Heme c since it is covalently bound to the protein. If I add hemin after overexpression, the protein might not bind to heme properrly? That's my guess. I am not sure if that is correct.