Figure 2

Purification of PsbS from inclusion bodies using nickel-affinity column or urea-wash protocol. (a) Inclusion bodies pellet P4 (lane 1) containing 19 mg of PsbS was solubilized in urea buffer containing 0.5% LDS. Pellet (lane 2) and supernatant (lane 3) were obtained after centrifuging at 10000xg for 10 minutes. Supernatant was loaded on the nickel affinity column. The loss fractions of PsbS are shown as the flow through (lane 4) and wash (lane 5,6), and together give a loss of ~14 mg PsbS; (b) Nickel-affinity column purified elution frations of PsbS (1 µl loaded on gel). Elution fraction 3 (0.7 µg/µl), fraction 4 (1 µg/µl) and fraction 5 (0.7 µg/µl) were further used to refold PsbS (lane 3,4,5). In total, the collected fractions contained ~3.6 mg of PsbS out of 19 mg starting material. (c) UP-wash protocol; Lane 1: About 3.2 mg of PsbS from the inclusion bodies pellet was dissolved in buffer containing 8 M urea. Several washes of urea buffer were carried out (Lane 2, 3, 4). Lane 5 is washing step of pellet with 8 M urea buffer with 0.05% LDS. The last wash step was carried out using urea buffer with 0.5% of LDS to dissolve all the PsbS from inclusion bodies (Lane 6). The concentration of PsbS in the 0.5% LDS buffer was ~2 µg/µL (2.5 µL sample loaded on gel). In total ~2 mg of PsbS was present after urea-wash purification from 3.2 mg starting material.