Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence.
A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.
Aliverti
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A
Dipartmento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Jansen
T
T
Zanetti
G
G
Ronchi
S
S
Herrmann
R G
RG
Curti
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis*; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
D003062:Codon; D004247:DNA; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases