Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli.
The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
M F
MF
Taylor
M H
MH
Boylan
D E
DE
Edmondson
D000595:Amino Acid Sequence; D001395:Azotobacter / Q000032:analysis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism; D005418:Flavodoxin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005420:Flavoproteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010710:Phosphates; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; D010710:Phosphates; D011994:Recombinant Proteins