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

doi:10.1006/bbrc.1993.2626

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

Medicine, Cardiology Division

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

Abstract

A synthetic gene encoding the peptide sequence for the low molecular weight (M_r ∼ 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 language	English (US)
Pages (from-to)	1357-1362
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	197
Issue number	3
DOIs	https://doi.org/10.1006/bbrc.1993.2626
State	Published - Dec 30 1993

ASJC Scopus subject areas

Molecular Biology
Biophysics
Biochemistry
Cell Biology

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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.",

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AU - Agarwal, A.

AU - Tan, J.

AU - Eren, M.

AU - Tevelev, A.

AU - Lui, S. M.

AU - Cowan, J. A.

PY - 1993/12/30

Y1 - 1993/12/30

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

Abstract

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