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. 2014 Nov 4;290(2):1066–1074. doi: 10.1074/jbc.M114.613091

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FIGURE 2. — In vitro reconstitution of R. sphaeroides Rubisco with purified components. A, time course of RsRbcL folding and assembly analyzed by native PAGE and immunoblotting against RbcL. GuHCl denatured RsRbcL was diluted into GroEL/ES containing buffer and incubated either in the absence of ATP but presence of RsRbcS (panel i) or presence of ATP (panels ii–iv) without RsRbcS (panel ii), with RsRbcS (panel iii), or RsRbcS added after GroEL/ES cycling was stopped with apyrase (Apy) (panel iv). B, solubility of RsRbcL from reactions (panel i) to (panel iii) analyzed by fractionation into total (T), soluble (S), and pellet (P) fractions by centrifugation, followed by SDS-PAGE and immunoblotting against RbcL. C, Rubisco activities in reconstitution reactions as described in A. See legend for sequence of addition. Activities are expressed as percent of native enzyme control (k_cat ∼2 CO₂ s⁻¹) (15). Error bars indicate S.D. of at least three independent experiments.