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

Expression system for CRD from mouse LSECtin A cDNA for mouse LSECtin was isolated by polymerase chain reaction amplification from a mouse liver cDNA library from BD Biosciences (Oxford, UK). Initially, an expression system using the ompA signal sequence was constructed by direct analogy with the method previously used for the human protein (Powlesland et al. 2008) . For more efficient expression, synthetic oligonucleotides were combined with restriction fragments from the cDNA to insert the region encoding the CRD into vector T5T (Eisenberg et al. 1990) , with codons for an initiator methionine followed by an alanine codon and then the cDNA sequence beginning at residue 161 of mouse LSECtin, which encodes the sequence SerCysGlu at the N-terminal end of the CRD. For expression of biotin-tagged protein, the C-terminal end of the cDNA was similarly modified by insertion of synthetic oligonucleotides so that the expression vector codes for a protein in which the C-terminal cysteine residue of LSECtin is followed by the sequence GlyLeuAsnAspIlePheGluAlaGlnLysIleGluTrpHisGlu (Schatz 1993) . All sequences were confirmed by sequencing on an Applied Biosystems 310 genetic analyzer.