Two rounds of screening for scFv binding to complexes containing biotinylated membrane proteins were followed by one round of functional AP-2 screening to yield a high frequency pool of clones capable of binding to complexes containing both biotinylated membrane proteins and AP-2

Two rounds of screening for scFv binding to complexes containing biotinylated membrane proteins were followed by one round of functional AP-2 screening to yield a high frequency pool of clones capable of binding to complexes containing both biotinylated membrane proteins and AP-2. (BBB) membrane protein complexes made up of AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes made up of AP-2 were recognized. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important functions in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought. Keywords: antibody discovery, membrane protein, protein-protein interactions, yeast surface display Introduction Yeast surface display (YSD) is usually a powerful tool for combinatorial library screening and has been used to discover and engineer antibodies against a wide range of targets (Boder and Wittrup, 1997; Feldhaus relevance of the target antigens. A nonimmune human single-chain INT-767 antibody (scFv) library was screened to enrich for scFv that binds to those plasma membrane protein complexes that are also associated with AP-2. Through this screening process, and a nonexhaustive sampling of the resultant enriched library, we have discovered novel scFvs that target intracellular accessory proteins known to be involved in endocytosis and membrane trafficking, validating the fYDIP approach as a means to isolate antibodies against desired membrane protein complexes. Methods Media, cells and plasmids strain EBY100 was utilized for scFv surface display. The na?ve human INT-767 scFv library (Feldhaus for INT-767 10?moments at 4C. The supernatant was recovered and added to an ultracentrifuge tube, and the tube was filled with buffer made up of 10?mM Tris-HCl pH?7.5, 250?mM sucrose and 50?mM NaCl. Membranes were pelleted by centrifugation at 200,000for 90?moments at 4C. The supernatant was discarded, and the pelleted plasma membranes were resuspended and solubilized in tris-buffered saline (TBS) made up of 1% TritonX-100 (TX-100, IB07100, IBI Scientific). All buffers contained 1 protease inhibitor cocktail (PIC, 11836170001, Roche) and 2?mM EDTA. Brain microvessel isolation and plasma membrane fractionation Microvessels were isolated from bovine or rat brains following the method of Lidinsky and Drewes (1983). Isolated capillaries were incubated with 5?mM sulfo-NHS-LC-biotin (PG82075, Thermo Fisher) for 2?hours at 4C to selectively tag membrane proteins with biotin. The reaction was quenched by addition of glycine to a final concentration of 100?mM and incubation for 10?minutes on ice. Endothelial plasma membranes were fractionated from your capillaries using a two-step hypotonic lysis INT-767 process as follows: incubation in (i) distilled water at 4C for 2?hours and (ii) 10?mM Tris-HCl pH?7.4 at 4C for 30?moments. After each lysis step, the capillaries were pelleted by centrifugation at 15,000??resulted in a supernatant made up of dispersed plasma membrane fragments INT-767 and a pellet made up of the capillary basement membranes. All buffers contained 1 protease inhibitor cocktail (PIC, 11836170001, Roche) and 2?mM EDTA. The supernatant portion is referred to as brain microvessel plasma membranes (BMPM). In a typical BMPM isolation ~?4.4?mg of BMPM proteins were recovered from one bovine brain and ~?1.6?mg were recovered from 10 rat brains. Membrane protein complexes were solubilized via the addition of TX-100 to a final concentration Klf4 of 1% v/v, and free biotin was added to a final concentration of 1 1?mM prior to fYDIP experiments as described below. fYDIP screening and individual clone assay fYDIP screening is a modification around the previously reported YDIP method (Cho and Shusta, 2010; Tillotson and dissolved in 3% ACN, 0.1% formic acid in water. Samples were analyzed using a Waters nanoAcquity UPLC system coupled to a Thermo Scientific Orbitrap Elite mass spectrometer. Peptides were loaded onto a 75?m inner diameter microcapillary column fabricated with an integrated emitter tip and packed with 15?cm of bridged ethylene cross C18 particles (1.7?m, 130??, Waters). Mobile phone phase A was composed of water, 5% DMSO.