Reassembly of Synechocystis sp. PCC 6803 F1-ATPase from its over-expressed subunits

AbstractSubunits α, β, and γ of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors. Overexpressed subunit β was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant α and γ subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the α and β subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits α, β, γ, δ and ϵ. The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP