High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7 |
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Authors: | Okai Masahiko Ohtsuka Jun Asano Atsuko Guo Linjun Miyakawa Takuya Miyazono Ken-ichi Nakamura Akira Okada Akitoshi Zheng Hai Kimura Kenzo Nagata Koji Tanokura Masaru |
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Institution: | Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. |
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Abstract: | Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) 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. |
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