I wish to recombinantly express a cytochrome reductase which contains both FMN and FAD. I was planning on seeing if improvements to protein quality arise in the presence of the riboflavin precursor and was wondering if anyone had any particular information regarding which form works best, neutral or phosphorylated.
Hi Jason,
are you using an E. coli expression system? E. coli is lacking a transport system for riboflavin so additions of riboflavin to the medium is unlikely to have a tremendous effect. However, it is cheap and non-toxic and you may add it up to its limits of solubility, which may cause some of the external riboflavin to enter the cell. The phosphorylated form FMN is quite expensive and likely to be dephosphorylated by extracellular phosphatases, so this will not help. If you want to do the experiment properly you may wish to use one of the strains developed by Thilo Mathes which have a heterologous riboflavin transporter integrated into the genome. The strains were published in these two papers: PMID: 24223875 and PMID: 19027027.
Best wishes Jürgen