Synthesis, Cloning and Expression of a Synthetic Gene for High Potential Iron Protein from Chromatium vinosum

Abstract

A synthetic gene encoding the peptide sequence for the low molecular weight (Mr ∼ 9600 Da) high-potential iron protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonudeotides 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-β-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.