Synthesis, cloning and expression of a synthetic gene for high potential iron protein from chromatium vinosum

A. Agarwal, J. Tan, M. Eren, A. Tevelev, S. M. Lui, J. A. Cowan

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1357-1362
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume197
Issue number3
DOIs
StatePublished - Dec 30 1993

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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