Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae

Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (ψ)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Ψ-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His6-tagged Yol066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yol066 protein is best at low temperature (18 °C) and IPTG concentration (50 μM) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E. coli culture.