Computational protocol: Plug-and-Play Pairing via Defined Divalent Streptavidins☆

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[…] SA, SAe, D, and Dd streptavidin variants were expressed in Escherichia coli and refolded from inclusion bodies by dilution into phosphate-buffered saline (PBS) as previously described . After refolding, the protein mixtures of SA/Dd or SAe/D were first purified on a 5-mL iminobiotin-Sepharose affinity column (Affiland, S.A.) using 50 mM sodium borate and 300 mM NaCl, pH 11.0, as the binding buffer and 20 mM KH2PO4, pH 2.2, as the elution buffer, with a 5-mL/min flow rate. The eluate was then exchanged into 20 mM Tris–HCl, pH 8.0, by dialysis and loaded onto a 1-mL Mono-Q column (GE Healthcare). The different tetramers were then isolated using a 100-column-volume (i.e., 100 mL) linear gradient of 0–1 M NaCl, collecting 1-mL fractions with a 1-mL/min flow rate. For larger-scale preparations, a 30-mL Q-Sepharose High-Performance column (GE Healthcare) and a 30-column-volume gradient from 0.15 to 0.4 M NaCl was run. Eluted fractions were concentrated to 5–10 mg/mL using a Vivaspin cutoff 30-kDa centrifugal concentrator (GE Healthcare), dialyzed thrice into PBS, and stored at − 80 °C. All purification steps were performed using an ÄKTA purifier 10 (GE Healthcare). Yield from refolding 1 L of biotin-binding subunit mixed with 1 L of non-binding subunit was approximately 9 mg of the relevant divalent and 12 mg of the monovalent streptavidin.Monovalent streptavidin (SAe1D3) can also be purified, if preferred, in a gravity-flow column. The mixed refold of SAe and D, prepared in 20 mM Tris–HCl, pH 8.0, as above, is loaded onto 1 mL Q-Sepharose High-Performance resin (GE Healthcare) in a Poly-Prep chromatography column (Bio-Rad) pre-equilibrated with binding buffer of 20 mM Tris–HCl, pH 8.0. The column is washed with 10 mL 20 mM Tris–HCl, pH 8.0, plus 0.15 M NaCl (to wash out D4 and impurities), followed by elution of SAe1D3 with 5 mL 20 mM Tris–HCl, pH 8.0, plus 0.25 M NaCl, collecting 1-mL fractions. The resin is regenerated by washing with 10 mL 20 mM Tris–HCl, pH 8.0, plus 2 M NaCl.Glutathione S-transferase-BirA (the plasmid was a kind gift from Chris O'Callaghan, University of Oxford) was expressed in E. coli and purified using glutathione-Sepharose as described previously . AP-Affibody was expressed in E. coli and purified using Ni-NTA (Qiagen) . Enzyme-mediated biotinylation was performed as described previously .Protein concentrations were determined from A280 via ProtParam. Concentrations of all streptavidin forms refer to the concentration of the monomer. pI was predicted using ProtParam. [...] Data were auto-indexed and integrated by the xia2 program upon collection . The cis- and 1,3 trans-divalent streptavidin structures were phased by molecular replacement using PDB 3RY1 (apo streptavidin) and further refined using the Phenix suite of programs implemented through a Python graphical user interface . The models were altered to better fit the electron density using Coot . Throughout the refinement of apo 1,3 trans-divalent, all data were included from 24.98 Å resolution to the highest limit (1.59 Å), and anisotropic temperature factors were refined. For the 1,3 trans-divalent streptavidin with bound biotin-4-fluorescein, all data from 49.06 Å resolution to the highest limit (2.26 Å) and isotropic temperature factors were refined. Similarly, for the cis-divalent structure, all data were included from 42.12 Å resolution to the highest limit (1.43 Å), and anisotropic temperature factors were refined. All models were continually evaluated with MolProbity .The refinement statistics and other structure factors for the two structures are shown in .Structures were visualized, and images for figures were prepared using PyMOL (Schrödinger, LLC). Electron density maps were visualized and images were prepared (contoured at 1 rmsd) with the CCP4mg program . Predicting the clash between cis-bound biotin-4-fluorescein molecules was done by overlaying the biotin-4-fluorescein-bound streptavidin monomer with the adjacent ligand-free monomer using PyMOL. […]

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