Hyperexpression of a synthetic gene encoding a high potential iron sulfur protein.
A gene encoding high potential iron sulfur protein (HiPIP) iso-1 from Ectothiorhodospira halophila was constructed in one step from long synthetic oligonucleotides. The gene was inserted into a phagemid vector from which the HiPIP was expressed as a fusion protein to > 10% of the soluble protein in Escherichia coli, demonstrating that a 4Fe-4S protein can be highly expressed in E. coli. The recombinant HiPIP was purified to apparent homogeneity by affinity chromatography followed by proteolytic removal of the leader sequence and anion exchange chromatography. Approximately 180 mg of HiPIP were purified from 10 l of cell culture. CD spectra of the oxidized and reduced forms of the protein and the 1H NMR spectrum of the oxidized protein are essentially identical to those of the wild type protein, indicating that the environment of the iron sulfur cluster in the two proteins is the same and thus that the recombinant protein is folded correctly. The reduction potential of the recombinant protein was determined to be 120 +/- 6 mV versus NHE (20 mM HEPES, 0.1 M sodium chloride, pH 7.0, 25 degrees C). This efficient heterologous expression of an HiPIP enables a systematic investigation of structure-function relationships in this class of iron sulfur proteins.
Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
L D
LD
Eltis
S G
SG
Iwagami
M
M
Smith
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D045322:Photosynthetic Reaction Center Complex Proteins; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D007506:Iron-Sulfur Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D011993:Recombinant Fusion Proteins; C007943:high potential iron-sulfur protein