Mouse LSECtin as a model for a human Ebola virus receptor

Pipirou, Zoi; Powlesland, Alex S; Steffen, Imke; Pöhlmann, Stefan; Taylor, Maureen E; Drickamer, Kurt

The CRD of LSECtin was expressed in Escherichia coli strain BL21(DE3) grown in the Luria-Bertani broth containing 50 µg mL −1 ampicillin. For addition of biotin, the cells were also transformed with plasmid birA, which encodes the gene for biotin ligase (Chapman-Smith and Cronan 1999) , and chloramphenicol at 20 µg mL −1 was included in the medium. For protein production, a 200 mL starter culture was diluted to 6 L and grown at 37°C to OD 550 of 0.7, at which point additions were made to achieve final concentrations of 100 µg mL −1 isopropyl-β-D-thiogalactoside and 12.5 µg mL −1 biotin. After further growth for 2.5 h at 37°C, bacteria were harvested by centrifugation for 15 min at 3000 × g, washed in 400 mL of 10 mM Tris-Cl, pH 7.8, and collected by centrifugation for 10 min at 6000 × g. The final pellet was resuspended in 200 mL of 10 mM Tris-Cl, pH 7.8, and sonicated at full power in a Branson Model 250 sonicator for 6 × 30 s, with cooling on ice between sonication steps. The insoluble inclusion bodies were collected by centrifugation for 15 min at 10,000 × g and dissolved in 100 mL of 6 M guanidine hydrochloride containing 100 mM Tris-Cl, pH 7.0, by brief sonication. Following addition of 10 µL of 2-mercaptoethanol, the mixture was stirred for 30 min at 4°C and centrifuged for 30 min at 10,000 × g. The supernatant was dialyzed against three changes of 2 L of loading buffer (150 mM NaCl, 25 mM CaCl 2 , 25 mM Tris-Cl, pH 7.8), centrifuged for 5 min at 8000 × g and 30 min at 1,00,000 × g, the supernatant was filtered through glass wool and applied to a 10 mL column of mannose-or fucose-Sepharose (Fornstedt and Porath 1975) .