Recombinant production and purification of the subunit c of chloroplast ATP synthase

Robert M. Lawrence, Benjamin Varco-Merth, Christopher J. Bley, Julian Chen, Petra Fromme

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

In chloroplasts, the multimeric ATP synthase produces the adenosine triphosphate (ATP) that is required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of a ring of c-subunits, which is imbedded in the thylakoid membrane. The rotation of this c-subunit ring is driven by the translocation of protons across this membrane, along an electrochemical gradient. The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring (c n) in the enzyme, which is organism dependent. Although this ratio is inherently related to the metabolism of the organism, the exact cause of the cn variability is not well understood. In order to investigate the factors that may contribute to this stoichiometric variation, we have developed a recombinant bacterial expression and column purification system for the c 1 monomeric subunit. Using a plasmid with a codon optimized gene insert, the hydrophobic c1 subunit is first expressed as a soluble MBP-c1 fusion protein, then cleaved from the maltose binding protein (MBP) and purified on a reversed phase column. This novel approach enables the soluble expression of an eukaryotic membrane protein in BL21 derivative Escherichia coli cells. We have obtained significant quantities of highly purified c1 subunit using these methods, and we have confirmed that the purified c1 has the correct alpha-helical secondary structure. This work will enable further investigation into the undefined factors that affect the c-ring stoichiometry and structure. The c-subunit chosen for this work is that of spinach (Spinacia oleracea) chloroplast ATP synthase.

Original languageEnglish (US)
Pages (from-to)15-24
Number of pages10
JournalProtein Expression and Purification
Volume76
Issue number1
DOIs
StatePublished - Mar 1 2011

Keywords

  • ATP synthase
  • Chloroplast
  • Maltose binding protein
  • Recombinant expression
  • Subunit III
  • Subunit c

ASJC Scopus subject areas

  • Biotechnology

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