High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7.
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.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.
Masahiko
M
Okai
Jun
J
Ohtsuka
Atsuko
A
Asano
Linjun
L
Guo
Takuya
T
Miyakawa
Ken-ichi
K
Miyazono
Akira
A
Nakamura
Akitoshi
A
Okada
Hai
H
Zheng
Kenzo
K
Kimura
Koji
K
Nagata
Masaru
M
Tanokura
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D011312:Pressure; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D038181:FMN Reductase