Cloning, Expression, and Purification of SV5 F-GCNt

The basic approaches for cloning, expression and purification of SV5 F-GCNt have been described for hPIV3 F (ref. 20 (link)). In brief, complementary DNA encoding a form of the SV5 (W3A strain) F protein (FR3) in which the furin cleavage site had been mutated to prevent intracellular processing^{41 (link)} was cloned into pMelBac (Invitrogen) by standard PCR protocols. A soluble form of F was generated that contained the honeybee melittin signal sequence in place of the F signal sequence and, at the C terminus, an isoleucine zipper domain (GCNt)^{23 (link),42 (link)} in heptad repeat phase with HRB, followed by a factor Xa cleavage site and a His₆ tag. The nucleotide sequence of the construct was obtained with a 3100-Avant sequencer (Applied Biosystems). Recombinant baculovirus was generated with a Bac-N-Blue transfection kit (Invitrogen). The secreted F-GCNt protein was purified by Co^{2 (link)}-affinity chromatography.