A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

David E. Gordon; Gwendolyn M. Jang; Mehdi Bouhaddou; Jiewei Xu; Kirsten Obernier; Matthew J O'Meara; Jeffrey Z. Guo; Danielle L. Swaney; Tia A. Tummino; Ruth Huttenhain; Robyn Kaake; Alicia L. Richards; Beril Tutuncuoglu; Helene Foussard; Jyoti Batra; Kelsey Haas; Maya Modak; Minkyu Kim; Paige Haas; Benjamin J. Polacco; Hannes Braberg; Jacqueline M. Fabius; Manon Eckhardt; Margaret Soucheray; Melanie Brewer; Merve Cakir; Michael J. McGregor; Qiongyu Li; Zun Zar Chi Naing; Yuan Zhou; Shiming Peng; Ilsa T. Kirby; James E. Melnyk; John S Chorba; Kevin Lou; Shizhong A. Dai; Wenqi Shen; Ying Shi; Ziyang Zhang; Inigo Barrio-Hernandez; Danish Memon; Claudia Hernandez-Armenta; Christopher J.P. Mathy; Tina Perica; Kala B. Pilla; Sai J. Ganesan; Daniel J. Saltzberg; Rakesh Ramachandran; Xi Liu; Sara B. Rosenthal; Lorenzo Calviello; Srivats Venkataramanan; Yizhu Lin; Stephanie A. Wankowicz; Markus Bohn; Phillip P. Sharp; Raphael Trenker; Janet M. Young; Devin A. Cavero; Joseph Hiatt; Theo Roth; Ujjwal Rathore; Advait Subramanian; Julia Noack; Mathieu Hubert; Ferdinand Roesch; Thomas Vallet; Björn Meyer; Kris M. White; Lisa Miorin; Oren S. Rosenberg; Kliment A. Verba; David Agard; Melanie Ott; Michael Emerman; Davide Ruggero; Adolfo Garcí-Sastre; Natalia Jura; Mark von Zastrow; Jack Taunton; Olivier Schwartz; Marco Vignuzzi; Christophe d'Enfert; Shaeri Mukherjee; Matt Jacobson; Harmit S. Malik; Danica G Fujimori; Trey Ideker; Charles S Craik; Stephen Floor; James S. Fraser; John Gross; Andrej Sali; Tanja Kortemme; Pedro Beltrao; Kevan Shokat; Brian K. Shoichet; Nevan J. Krogan

Affinity purification. Frozen cell pellets were thawed on ice for 15-20 minutes and suspended in 1 ml Lysis Buffer [IP Buffer (50 mM Tris-HCl, pH 7.4 at 4°C, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Nonidet P 40 Substitute (NP40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche)]. Samples were then frozen on dry ice for 10-20 minutes and partially thawed at 37°C before incubation on a tube rotator for 30 minutes at 4°C and centrifugation at 13,000 xg, 4°C for 15 minutes to pellet debris. After reserving 50 μl lysate, up to 48 samples were arrayed into a 96-well Deepwell plate for affinity purification on the KingFisher Flex Purification System (Thermo Scientific) as follows: MagStrep "type3" beads (30 μl; IBA Lifesciences) were equilibrated twice with 1 ml Wash Buffer (IP Buffer supplemented with 0.05% NP40) and incubated with 0.95 ml lysate for 2 hours. Beads were washed three times with 1 ml Wash Buffer and then once with 1 ml IP Buffer. To directly digest bead-bound proteins as well as elute proteins with biotin, beads were manually suspended in IP Buffer and divided in half before transferring to 50 μl Denaturation-Reduction Buffer (2 M urea, 50 mM Tris-HCl pH 8.0, 1 mM DTT) and 50 μl 1x Buffer BXT (IBA Lifesciences) dispensed into a single 96-well KF microtiter plate, respectively. Purified proteins were first eluted at room temperature for 30 minutes with constant shaking at 1,100 rpm on a ThermoMixer C incubator. After removing eluates, on-bead digestion proceeded (below). Strep-tagged protein expression in lysates and enrichment in eluates were assessed by western blot and silver stain, respectively. The KingFisher Flex Purification System was placed in the cold room and allowed to equilibrate to 4°C overnight before use. All automated protocol steps were performed using the slow mix speed and the following mix times: 30 seconds for equilibration/wash steps, 2 hours for binding, and 1 minute for final bead release. Three 10 second bead collection times were used between all steps.