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.
Spinach ferredoxin I: overproduction in Escherichia coli and purification / L. Piubelli, A. Aliverti, F. Bellintani, G. Zanetti. - In: PROTEIN EXPRESSION AND PURIFICATION. - ISSN 1046-5928. - 6:3(1995 Jun), pp. 298-304.
|Titolo:||Spinach ferredoxin I: overproduction in Escherichia coli and purification|
ALIVERTI, ALESSANDRO (Secondo)
ZANETTI, GIULIANA (Ultimo)
|Parole Chiave:||ferredoxin ; iron-sulfur protein ; iron-sulfur cluster ; protein isoform ; electron transfer ; photosynthesis ; biological oxidoreduction ; recombinant protein|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore BIO/11 - Biologia Molecolare
|Data di pubblicazione:||giu-1995|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1006/prep.1995.1039|
|Appare nelle tipologie:||01 - Articolo su periodico|