S transformed into BL21(DE3) cells and expressed alone. Protein expression was induced at culture OD600 = 0.6?.8 with 0.5 mM IPTG and conducted at 16uC for 18 h. Cells were harvested by centrifugation, resuspended in 15 ml lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl) per L of culture, and mixed together prior to treatment with lysozyme (5 mg per L of culture), Complete Protease Inhibitor Tablet (Roche), 1 mM PMSF. Cells were then sonicated, and lysates treated with DNaseI, clarified by centrifugation and filtration, and supplemented with 1 mM DTT and 0.1 Triton-X 100.Figure 1. The IPP complex forms a stable, monodisperse, heterotrimeric complex. A) Schematic diagram of the IPP complex: Integrin-linked kinase (ILK; magenta), PINCH (green) and Parvin (blue). ILK is the hub of the complex, and binds the LIM1 domain of PINCH-1 via its N-terminal ankyrin-repeat domain (ARD), and the C-terminal calponin homology (CH2) domain of a-parvin via its C-terminal pseudokinase domain (pKD) to form the IPPmin complex. The 14 residue inter-domain linker in ILK is shown. The lengths of the proteinsProtein PurificationLysates were applied to glutathione-agarose 4B beads (GE Healthcare) at 4uC and MedChemExpress IQ-1 collected by gravity flow. The flowSAXS Analysis of the IPP ComplexFigure 2. SAXS analysis for IPPmin reveals a globular heterotrimeric complex. A) SAXS intensity profiles (logarithmic) for four concentrations of the IPPmin complex. B) Linearity of Guinier plots with manual selection of Guinier region. The Rg values are presented in Table 1. Automatic Guinier analysis performed in AutoRG [29], which is ASP015K manufacturer consistent with the analysis shown here, is presented in the Supporting Information. C) Normalized pair distribution functions P(R) calculated automatically with AutoGNOM [30]. D) Dimensionless Kratky plots support a globular shape. doi:10.1371/journal.pone.0055591.gthrough sample was collected, and reapplied to the glutathione column a total of three times. The beads were washed three times with 10 column volumes (CV) of lysis buffer plus 1 mM DTT, and the column flow stopped before addition of freshly prepared elution buffer (15 mM reduced glutathione in lysis buffer, 1 mM DTT). Beads were incubated with elution buffer for 5 minutes, and the eluate collected. Elution was performed with 7?0 fractions of elution buffer, and the evaluated by SDS-PAGE. Elution fractions containing IPP complex were pooled. His-tagged recombinant 18325633 TEV protease was added at a final concentration of 0.01?.1 mg/ml and incubated overnight at 4uC, to remove the GST- and (His)-tags. The sample was then diluted for injection onto a 1 mL Mono Q column (GE Healthcare) to 50 mM Tris, pH 7.5, 30 mM NaCl, 1 mM DTT. A shallow gradient over 80 CV from 3 to 13 Buffer B (50 mM Tris pH 7.5, 1 M NaCl, 1 mM DTT) was applied in order to differentially elute GST from IPP protein, and 2 ml fractions collected. To remove remaining contaminating (His)-TEV protease and/or GST, the fractions containing IPP complex proteins (as determined by SDS-PAGE) were incubated with 50 ml of glutathione-agarose 4B plus 50 ml NiAgarose beads for 1 h at 4uC. The sample was then concentrated to 2 ml in a Centrifugal Filtration Unit (Millipore) and further purified by size-exclusion chromatography (Superdex 200 prep grade 16/60; GE Healthcare) equilibrated in 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT. Fractions containing IPP proteins were pooled and concentrated to a final concentration of 7.0 mg/ml and filtered through a 0.S transformed into BL21(DE3) cells and expressed alone. Protein expression was induced at culture OD600 = 0.6?.8 with 0.5 mM IPTG and conducted at 16uC for 18 h. Cells were harvested by centrifugation, resuspended in 15 ml lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl) per L of culture, and mixed together prior to treatment with lysozyme (5 mg per L of culture), Complete Protease Inhibitor Tablet (Roche), 1 mM PMSF. Cells were then sonicated, and lysates treated with DNaseI, clarified by centrifugation and filtration, and supplemented with 1 mM DTT and 0.1 Triton-X 100.Figure 1. The IPP complex forms a stable, monodisperse, heterotrimeric complex. A) Schematic diagram of the IPP complex: Integrin-linked kinase (ILK; magenta), PINCH (green) and Parvin (blue). ILK is the hub of the complex, and binds the LIM1 domain of PINCH-1 via its N-terminal ankyrin-repeat domain (ARD), and the C-terminal calponin homology (CH2) domain of a-parvin via its C-terminal pseudokinase domain (pKD) to form the IPPmin complex. The 14 residue inter-domain linker in ILK is shown. The lengths of the proteinsProtein PurificationLysates were applied to glutathione-agarose 4B beads (GE Healthcare) at 4uC and collected by gravity flow. The flowSAXS Analysis of the IPP ComplexFigure 2. SAXS analysis for IPPmin reveals a globular heterotrimeric complex. A) SAXS intensity profiles (logarithmic) for four concentrations of the IPPmin complex. B) Linearity of Guinier plots with manual selection of Guinier region. The Rg values are presented in Table 1. Automatic Guinier analysis performed in AutoRG [29], which is consistent with the analysis shown here, is presented in the Supporting Information. C) Normalized pair distribution functions P(R) calculated automatically with AutoGNOM [30]. D) Dimensionless Kratky plots support a globular shape. doi:10.1371/journal.pone.0055591.gthrough sample was collected, and reapplied to the glutathione column a total of three times. The beads were washed three times with 10 column volumes (CV) of lysis buffer plus 1 mM DTT, and the column flow stopped before addition of freshly prepared elution buffer (15 mM reduced glutathione in lysis buffer, 1 mM DTT). Beads were incubated with elution buffer for 5 minutes, and the eluate collected. Elution was performed with 7?0 fractions of elution buffer, and the evaluated by SDS-PAGE. Elution fractions containing IPP complex were pooled. His-tagged recombinant 18325633 TEV protease was added at a final concentration of 0.01?.1 mg/ml and incubated overnight at 4uC, to remove the GST- and (His)-tags. The sample was then diluted for injection onto a 1 mL Mono Q column (GE Healthcare) to 50 mM Tris, pH 7.5, 30 mM NaCl, 1 mM DTT. A shallow gradient over 80 CV from 3 to 13 Buffer B (50 mM Tris pH 7.5, 1 M NaCl, 1 mM DTT) was applied in order to differentially elute GST from IPP protein, and 2 ml fractions collected. To remove remaining contaminating (His)-TEV protease and/or GST, the fractions containing IPP complex proteins (as determined by SDS-PAGE) were incubated with 50 ml of glutathione-agarose 4B plus 50 ml NiAgarose beads for 1 h at 4uC. The sample was then concentrated to 2 ml in a Centrifugal Filtration Unit (Millipore) and further purified by size-exclusion chromatography (Superdex 200 prep grade 16/60; GE Healthcare) equilibrated in 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT. Fractions containing IPP proteins were pooled and concentrated to a final concentration of 7.0 mg/ml and filtered through a 0.