Fig.1

Design of a recombinant protein tag for efficient biotinylation and purification of monomeric proteins for capture on streptavidin-coated surfaces. (A) A schematic diagram showing the sequences of the C-terminal tags used in this study. A cartoon of a typical membrane-embedded type I cell surface receptor is shown next to the corresponding soluble ectodomain expressed as a bait. Lollipops represent potential N-linked glycosylation sites. The sequences of the C-terminal tags are shown and named. “LN” marked in black is the C-terminal end of the Cd4 tag. The peptide sequence that is a substrate for the BirA enzyme is highlighted in red, and the biotinylatable lysine residue is underlined. Flexible linkers and structurally insulating sequences are shown in purple and blue respectively, and oligo-histidine is shown in green. (B) Comparison of the biotinylation efficiencies of a rat Cd200R bait protein containing different C-terminal tags. The different Cd200R bait proteins were expressed with the modified BirA plasmid, normalized to 1 μg/ml, serially diluted on streptavidin-coated microtiter plates, and quantified by ELISA. (C) Cd200R-Bio-l-His bait proteins were cotransfected with a codon-optimized Flag-tagged BirA enzyme (BirA–Flag) or a His8-tagged non-codon-optimized BirA (BirA–His8). Bait expression levels were normalized to 1 μg/ml, and the relative fraction that was biotinylated was determined by ELISA after capture on a streptavidin-coated microtiter plate. (D) Bait proteins containing BLH fusion tag can be efficiently purified on Ni²⁺–NTA resin. Rat Cd200R–BLH baits were expressed and dialyzed to remove free biotin (to enable capture of unpurified supernatants) and then purified using Ni²⁺–NTA resin. Essentially all Cd200R–BLH protein was captured by the Ni²⁺–NTA resin (cf. before and after columns) and was efficiently eluted; little Cd200R–BLH protein leached during washing. Data points in panels B to D represent means ± standard errors (n ⩾ 3).