Thermolysin from bacillus thermoproteolyticus

Thermolysin from Bacillus thermoproteolyticus (Sigma–Aldrich) was dissolved in 45% DMSO, 0.05 M MES pH 6.0 at a concentration of 100 mg ml⁻¹. Crystals were grown using the hanging-drop vapour-diffusion method, where the drops were composed of the protein solution and a solution consisting of 0.05 M MES pH 6.0, 1 M NaCl, 45% DMSO in a 1:2 ratio. The reservoir contained 35% ammonium sulfate. Crystals were cryoprotected by transferring them for 5 s into a drop containing 6 M trimethylamine N-oxide.

Tubeimosides I (Cas No. 102040-03-9), Tubeimosides II (Cas No. 115810-12-3), and Tubeimosides III (Cas No. 115810-13-4) were purchased from Chengdu Biopurity Phytochemicals Ltd, China; U18666A (Cat No. 50205-0551) was from Cell Signaling Technology; Cathepsin B substrate (Cat No. sc-215529) and Cathepsin L substrate (Cat No. sc-3136) were from Santa Cruz Biotechnology; Filipin III (Cat No. GC12048) and CA074 (Cat No. GC15917) were from GLPBIO; EIPA (Cat No. HY-101840) and EST (E-64-d) (Cat No. HY-100229), and cholesterol (Cat No. HY-N0322) were from MedChemExpress; N-Dodecyl-β-D-maltoside (DDM) (Cat No. D4641), recombinant soluble ACE2 (Cat No. SAE0064), thermolysin from Bacillus thermoproteolyticus (Cat No. P1512), and anti-protease inhibitor cocktail (Cat No. P8340) were from Sigma Aldrich; Lyso-Tracker Red (Cat No. C1046), phosphoramidon (Metalloproteinase inhibitor) (Cat No. SG2024), and acetate buffer pH5.2 (Cat No. ST351) were from Beyotime Biotechnology; Lipofectamine 3000 (Cat No. L3000015) and Blasticidin S HCl (R21001) were from ThermoFisher; Puromycin (Cat No. ant-pr-1) was from InvivoGen; polyethyleneimine (Cat No. 23966-1) was from Polysciences, Inc; recombinant NPC1-C protein (Cat No. 16499-H32H) and recombinant SARS-CoV-2 spike RBD protein (Cat. No. 40592-V08H) were from SinoBiological.