The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts.
The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.
Serra
E C
EC
Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Krapp
A R
AR
Ottado
J
J
Feldman
M F
MF
Ceccarelli
E A
EA
Carrillo
N
N
D001483:Base Sequence; D001693:Biological Transport, Active; D002736:Chloroplasts / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D018744:DNA, Plant; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005287:Ferredoxin-NADP Reductase