Cloning, Expression, and Crystallization of the Fusion Protein of Newcastle Disease Virus

AbstractWe have recently reported the X-ray crystal structure of a fragment of the fusion protein (F) of Newcastle disease virus (NDV). This work describes the methodology involved in the production and crystallization of that protein in recombinant form. The full-length cDNA of NDV-F was cloned and the ectodomain expressed in both CHO-K1 and Lec-3.2.8.1 cells. The recombinant protein, secreted as a single-chain polypeptide F0′, was purified using a c-myc antibody affinity column followed by gel filtration chromatography. Electron microscopic imaging showed the F0′ product to consist of unaggregated club-shaped particles. Trypsin treatment of F0′ could be used to produce disulfide-linked F2 and F1′ chains. However, imaging revealed extensive rosette-like aggregation of the trypsin-treated material, indicative of a conformational change. Only the non-trypsin-treated product was thus suitable for crystallization and two crystal forms were obtained, diffracting to ca. 3.5 and 4.0 Å, respectively. Both crystal forms were used in the structure determination