Purification and biotinylation of GFP nanobody

The construct for a recombinant GFP nanobody (clone LaG-16; Fridy et al., 2014) was a kind gift from Dr. Michael Rout. The plasmid was transformed into BL21-CodonPlus (DE3)-RIL (Strategene) cells, and periplasmic overexpression was induced with 0.1 mM IPTG for 16 h at 16°C. Cells from 6 liters of culture were harvested at 5,000 g for 10 min and resuspended in TES buffer (200 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 500 mM sucrose). The cells were osmotically shocked on ice for 30 min by diluting them fivefold in 1:4 water/TES buffer. Periplasmic extract was separated from cell debris by centrifugation for 10 min at 6,000 g, and this supernatant was further clarified at 20,000 g for 20 min at 4°C. The final supernatant was supplemented with NaCl to 150 mM and incubated with 3 ml His60 resin (Takara Biosciences) for 30 min. The resin was washed in batch with wash buffer 1 (20 mM Tris-HCl, pH 8.0, and 900 mM NaCl) and wash buffer 2 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10 mM imidazole) and loaded onto a disposable column, and the protein was eluted with Ni-NTA elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 250 mM imidazole). Peak fractions were identified by Bradford assay, pooled, and concentrated with a 10-kD cut-off spin filter (Millipore). To produce biotinylated GFP nanobody, N-hydroxysuccinimide (NHS) esters of biotin ((NHS)-biotin; Millipore Sigma; H1759-5MG) was dissolved in DMSO and added to the concentrated protein at a molar ratio of 10:1 such that the final DMSO concentration did not exceed 5%. The reaction was incubated at 4°C for 4 h and clarified by centrifugation at 21,000 g for 20 min at 4°C. The nanobody was then gel-filtered over a Superdex 75 10/300 column (Cytiva) equilibrated in coupling buffer (150 mM sodium bicarbonate, pH 8.0, and 150 mM NaCl). Biotinylated antibody was supplemented with glycerol to 10% (vol/vol), flash-frozen in liquid N₂, and stored at −80°C.