Heterologous Overexpression of Human FAD Synthase Isoforms 1 and 2

The study of human FAD synthase enzymes requires a recombinant strategy to produce large amount of purified proteins in a soluble form. E. coli was exploited to this aim. To achieve the production of FAD synthase in a large scale, E. coli strains, plasmids (promoter, tags), growth temperature, inducer concentration, medium composition, and osmotic pressure were optimized. To date there is no universal protocol for protein expression, but for each protein a specific combination of “expression parameters” can be selected in order to maximize the results. An experimental protocol for the expression of two isoforms of the human FAD synthase was set up. The final procedures are based on the use of E. coli Rosetta(DE3) strain. Two different plasmids were used to obtain optimal amount of the two protein isoforms. In both cases, following the addition of the IPTG inducer, the growth temperature was lowered to increase the solubility of the recombinant protein. The detailed procedures for FAD synthase isoform 1 and isoform 2 overproduction are described in this protocol.