Spinach ferredoxin i: overproduction in Escherichia coli and purification.
Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.
Piubelli
L
L
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Aliverti
A
A
Bellintani
F
F
Zanetti
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000737:chemistry* / Q000235:genetics; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018724:Spinacia oleracea / Q000737:chemistry* / Q000235:genetics
D005288:Ferredoxins; D011994:Recombinant Proteins