The sequence encoding residues 1-86 of pr was amplified by PCR of an expression plasmid for DENV2 NGC prM-E DI/II [30] . The PCR product was ligated into the pPUR vector (Clontech), with the 21-residue TPA signal peptide [65] fused at the N-terminus and a tandem Strep tag at the C terminus (Fig. 1) . The plasmid, referred to as pPUR-TPA-pr-STST, was transfected into 293T cells using polyethylenimine (PEI, Polysciences). For optimal protein production, 3.5610 6 cells were plated per 10 cm dish and cultured for 24 h in 10 ml of complete medium. 7.5 mg plasmid in 1 ml DME was mixed with 30 mg PEI, incubated 10 min, then added drop wise to the cell culture medium. After 12 h, the medium was changed to 10 ml DME plus 2% serum. The culture medium was collected after 48h and again after 72h. Pr was purified by affinity chromatography on a Strep-Tactin column from IBA BioTAGnology and by gel filtration using a Sephadex G75 column [30] . Final yields were ,2 mg purified protein/1 liter culture supernatant.