Spinach ferredoxin i: overproduction in Escherichia coli and purification

Protein Expr Purif. 1995 Jun;6(3):298-304. doi: 10.1006/prep.1995.1039.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chloroplasts / chemistry*
  • Chloroplasts / genetics
  • Chromatography, Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Ferredoxins / biosynthesis
  • Ferredoxins / genetics
  • Ferredoxins / isolation & purification*
  • Mass Spectrometry
  • Molecular Sequence Data
  • Protein Engineering
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Spinacia oleracea / chemistry*
  • Spinacia oleracea / genetics

Substances

  • Ferredoxins
  • Recombinant Proteins