Recombinant production and purification of the subunit c of chloroplast ATP synthase.
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 c(n) 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 c₁ 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.
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Robert M
RM
Lawrence
Benjamin
B
Varco-Merth
Christopher J
CJ
Bley
Julian J-L
JJ
Chen
Petra
P
Fromme
D025222:Chloroplast Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D018724:Spinacia oleracea / Q000201:enzymology
D058619:Maltose-Binding Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D025222:Chloroplast Proton-Translocating ATPases