In order to express soluble trimeric subtype 5 HA (sH5 3 ) in mammalian cells, the H5 ectodomain-coding sequence was first cloned into an appropriate expression vector. In the pCD5 vector used, the H5-sequence was preceded by a signal peptide-encoding sequence, to allow efficient secretion of the recombinant protein, and followed by sequences coding for the GCN4 isoleucine-zipper trimerizaton motif [21] and the Strep-tag II, the latter for purification purposes (Fig. 1A) . Expression of the H5 ectodomain was achieved by transient transfection of the expression plasmid into HEK cells. Expression and secretion of the H5 protein was verified by subjecting cell culture supernatant to gel electrophoresis followed by western blotting using an antibody directed against Strep-tag II (Fig. 1B) . The results show that the recombinant H5 protein could be readily detected in the cell culture supernatant after transfection of the cells with the expression plasmid, but not after mock transfection. The secreted H5 protein was purified in a single step protocol by using Streptactin sepharose beads. Protein yields varied between 0.4-1 mg of recombinant protein per 100 ml cell culture medium. After purification of the H5 protein from the cell culture supernatant, the oligomeric state of the H5 protein was analyzed by gel filtration column chromatography (Fig. 1C) . The bulk of the H5 protein eluted with the velocity of an oligomer, while only a minor fraction was found as aggregates in the void volume. The trimeric nature of the H5 oligomer was confirmed using blue-native gel electrophoresis followed by western blotting (Fig. 1D) . When the H5 preparation was heat-denatured for increasing time periods prior to electrophoresis, the initially trimeric HA species dissociated into dimers and monomers. The biological activity of the purified sH5 3 was studied using a solid phase-binding assay with the sialylated blood glycoprotein fetuin. Binding of sH5 3 was measured by means of the HRP conjugated to the anti-Strep-tag II antibody as detailed in the Material and Methods section. The H5 preparation exhibited a concentration dependent binding to the fetuin. This binding was sialic acid-dependent, as no binding was observed when the fetuin had been treated with neuraminidase (VCNA; Fig. 1E ). In conclusion, biologically active sH5 3 was efficiently produced using a mammalian expression system and readily purified.