Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi.
Photobacterium leiognathi lumazine protein has been expressed in Escherichia coli in high yield, 30 mg/l. The cloned gene was one previously reported by Illarionov (EMBL X56534), that had a similar sequence and was located in the same position as the lumazine protein gene in P. phosphoreum. This gene was placed downstream of the T7 gene 10 promoter of the plasmid pT7-7. When the E. coli are grown at 37 degrees C the protein accumulates in inclusion bodies but solubilization can be achieved in 6 M urea. By a simple procedure of dialysis in the presence of riboflavin and centrifugation, without any chromatography, the recombinant holoprotein is purified to 95% homogeneity. The spectral properties of this recombinant riboflavin protein are the same as those of a fluorescent riboflavin-bound protein produced by many strains of P. leiognathi. The absorption spectrum has the same maxima, 276, 386, 464 nm, the circular dichroism is also the same, and both absorption spectrum and CD are the same as that of apo-lumazine protein having riboflavin bound. The riboflavin on the recombinant can be easily replaced by 6,7-dimethyl-8-ribityllumazine. The absorption and fluorescence spectra, fluorescence yield, and bioluminescence properties of this rebound protein identify it as authentic lumazine protein.
Illarionov
B
B
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Illarionova
V
V
Lee
J
J
van Dongen
W
W
Vervoort
J
J
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012256:Riboflavin / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C026648:lumazine protein, Photobacterium; D012256:Riboflavin