Synthesis, cloning and expression of a synthetic gene for high potential iron protein from Chromatium vinosum.
A synthetic gene encoding the peptide sequence for the low molecular weight (M(r) approximately 9600 Da) high-potential iron protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonucleotides varying in size from 42-mers to 48-mers. After cloning the gene into a pET-21d(+) vector, expression of holoprotein in yields of 35 mg/liter of culture was obtained following induction with isopropyl-beta-D-thiogalactoside (IPTG). The recombinant protein was characterized by electronic absorption, 1H NMR, electrochemistry, N-terminal sequencing and amino acid analysis. This is the first example of the expression of a high potential ferredoxin containing a fully constituted [Fe4S4] cluster.
Agarwal
A
A
Evans Laboratory of Chemistry, Ohio State University, Columbus 43210.
Tan
J
J
Eren
M
M
Tevelev
A
A
Lui
S M
SM
Cowan
J A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D002844:Chromatium / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005288:Ferredoxins / Q000096:biosynthesis*; D015870:Gene Expression; D005813:Genes, Synthetic; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping
D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D009838:Oligodeoxyribonucleotides; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; C007943:high potential iron-sulfur protein