CLONING AND EXPRESSION OF THE GENE ENCODING FLAVODOXIN FROM DESULFOVIBRIO-VULGARIS (MIYAZAKI-F)

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli, A 1.6-kbp DNA fragm ent, isolated from D, vulgaris (Miyazaki F) by double digestion with S ail and EcoRI, contained the flavodoxin gene and its regulatory region , An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E, coli, The purified protein was soluble and exhibited a characteristic visible absorption spectrum, HPLC anal ysis of the recombinant flavodoxin revealed the presence of an identic al FMN to that found in the native D, vulgaris flavodoxin, and its dis sociation constant with FMN was determined to be 0.38 nM, In vitro H-2 reduction analysis indicated that the recombinant flavodoxin is activ e, and its redox potential was determined to be E-1 = -434 and E-2 = - 151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respect ively. Its redox behavior was also examined with the recombinant flavo doxin adsorbed onto a graphite electrode. The mutant, A16E, was also p roduced, which revealed time feature of a conserved Glu residue at the surface of the molecule.