Figure 5. QC photocleavage as a method for protein purification.

A) Schematic showing general protein purification procedure using QC-BSA and cell lysate. QC-BSA in a mixture with cell lysate was incubated with 8 for 14 h in the dark and subsequently quenched with QCOAc. The samples were transferred to Amicon Ultra centrifugal filters and washed with PBS three times. The concentrated samples were incubated with Pierce™ Streptavidin Agarose for 30 min. The resin was washed with PBS by spin filtration seven times (Washes 1-7). Following the washes, the resin was suspended in PBS and irradiated with 365 nm light by a handheld lamp for 5 min and the supernatant was eluted (Elution 1). The irradiation was repeated three more times with additional PBS each time (Elutions 2-4). The beads were then stripped of all remaining protein with 8 M guanidine HCl (pH = 1.5, 4 washes) and this was concentrated on an Amicon Ultra centrifugal filter (Elution B). Washes 1; 2,3; 6,7 and Elutions 1; 2; B were separated by gel electrophoresis and visualized with Coomassie staining. B) The general procedure from Figure 5A was followed with the following quantities of reagents: QC-BSA or BSA (100 μL in PBS, 1 mg/mL), 8 (100 μL, 50 μM in PBS), QCOAc (2.5 μL, 200 mM in DMSO), PBS rinse (200 μL, then twice with 350 μL), Pierce Streptavidin Agarose (100 μL), PBS washes (50 μL each, lanes Wash 1; 2,3; 6,7), elutions (50 μL PBS each, lanes Elution 1 and 2), guanidine HCl (50 μL washes, Elution B). C) Protein from Wash 1 and Elution 1 of BSA and QC-BSA samples from Figure 5B were subjected to ESI intact mass analysis. Unmodified BSA: BSA with no QC modifications. The numbers represent the number of QC moieties attached to BSA. D) The general procedure from Figure 5A and 5B was performed in the presence of bacterial cell lysate (10 μL, 4.94 g/mL) and K₃Fe(CN)₆ (10 μL, 22 mM). K₃Fe(CN)₆ and lysate were added sequentially to the solutions of QC-BSA or BSA before addition of 8. After QC ligation with 8, the capture, wash, and elutions sequence depicted in Figure 5A was performed. Lanes are as described in Figure 5B.