High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7

Flavin reductase HpaCSt catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaCSt, which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaCSt was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS–PAGE. The purified refolded HpaCSt did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.

► High pressure was used for refolding of flavin reductase HpaCSt.
► The refolded HpaCSt did not contain FMN.
► Crystals of the refolded HpaCSt were obtained without and with flavin compound.