Computational protocol: Structure-guided Discovery of Dual-recognition Chemibodies

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[…] Rat DPP-IV enzyme and Fab mutant proteins were expressed and purified as previously described. The purified DPP-IV/Fab 11A19-sitagliptin hybrid complex was concentrated to 6.6 mg/ml in buffer containing 20 mM Tris 7.9 and 200 mM NaCl. Crystals were grown at 20 °C in sitting drop trays with 0.1 µl protein solution plus 0.1 µl reservoir solution of 22% PEG 1000 and 0.1 M sodium citrate tribasic dihydrate pH 5.5. For x-ray diffraction data collection, crystals were transferred into well solution with 30% (w/v) ethylene glycol and then flash frozen in liquid nitrogen.X-ray diffraction data sets were collected at beamline 502 at Advanced Light Source (ALS) (Berkeley, CA) and processed with programs MOSFLM and SCALA in the CCP4 program suite. The structure was solved to a resolution of 2.77 Å by the molecular replacement program Phaser using a previously reported complex structure of rat DPP-IV with Fab 11A19 (PDB code: 4FFV) as a search model. Model building and refinement were carried out in COOT and REFMAC in CCP4, respectively. All structural figures were prepared using PyMOL (Schrodinger Inc, San Diego, CA). […]

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