Cloning, expression, and crystallization of the fusion protein of Newcastle disease virus.
We 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 A, respectively. Both crystal forms were used in the structure determination.
Chen
L
L
Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia.
Colman
P M
PM
Cosgrove
L J
LJ
Lawrence
M C
MC
Lawrence
L J
LJ
Tulloch
P A
PA
Gorman
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D008854:Microscopy, Electron / Q000379:methods; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins