Phage Display Selections for Affinity Reagents to Membrane Proteins in Nanodiscs

Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

“…The phage display sorting procedure performed to generate the sABs (Figure 1A) is based on a previously described solution capture scheme utilizing a Kingfisher magnetic beads handler (Fellouse et al, 2007; Paduch et al, 2013; Dominik and Kossiakoff, 2015). Membrane proteins were reconstituted into nanodiscs (Figures 1B and 1C) after optimization following established protocols (Ritchie et al, 2009; Bayburt and Sligar, 2010).…”

“…Prior to library sorting, the efficiency of biotinylation of antigens was evaluated by pull-down on streptavidin-coated magnetic beads (Streptavidin MagneSpehere Paramagnetic particles, Promega). Library sorting steps were performed using sAB Library E (kindly provided by S. Koide (Miller et al, 2012)) based on described protocols (Fellouse et al, 2007; Koide et al, 2009; Paduch et al, 2013; Dominik and Kossiakoff, 2015) with following modifications: 1) five, instead of four, rounds of library sorting were performed, 2) experiments with nanodiscs were performed in either buffer B (1% BSA in buffer A) or buffer C (1 mM EDTA in buffer B for CorA wild-type), while experiments with Mj 0480 in detergent – in buffer D (buffer B with 0.05% DDM), 3) for experiments with nanodiscs, starting from 2 nd sorting round, non-biotinylated empty nanodiscs (and, where indicated, CorA D253K nanodiscs) were used as soluble competitors, 4) elution of phage particles in experiments with nanodiscs and detergent were achieved by incubation in buffer B supplemented with 1% FC-12 and 100 mM DDT, respectively. The details of the strategy along with concentrations of membrane protein antigens throughout the library sorting experiments are further described in the Supplemental Information.…”

“…Single-point phage ELISA was used in the primary validation of binding affinities of generated sABs in phage format as described previously (Dominik and Kossiakoff, 2015). Amplified phage particles at 10-fold dilution were assayed against 20 nM biotinylated membrane proteins in either nanodisc or detergent using HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare, #27-9421-01).…”

“…These methods with several modifications have also proven successful for phage display selections to generate sABs to membrane proteins in detergent (Uysal et al, 2009; Kim et al, 2011; Li et al, 2014). However, our experience has shown that for membrane proteins, the process is not straightforward and the required adaptations are system dependent (Dominik and Kossiakoff, 2015). In particular, in many cases membrane protein stability is compromised by the detergent resulting in structural heterogeneity that confounds the generation of sAB binders to a single conformational state (unpublished data).…”

Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins

“…The optimal molar ratio of the components used in the reconstitution of the protein in nanodiscs is a 1:5:250 ratio of MalFGK2:MSP:lipids. The empty nanodiscs were separated from the loaded ones by IMAC following published protocols 32,33 . The transporter was further purified by SEC on a 10/300 S200 column to obtain monodispersed sample for activity assay.…”