Purification and analysis of a polydnavirus gene product expressed using a poly-histidine baculovirus vector.
The VHv1.1 polydnavirus gene has been implicated in suppressing the encapsulation response in parasitized insects [Li and Webb (1994) J. Virol. 68, 7482-7489]. In order to characterize this gene product and to further our analysis of its immunosuppressive function, we expressed the VHv1.1 using a custom-designed C-terminal poly-histidine baculovirus vector which allows for high expression and single-step purification of the protein. The 34 kDa VHv1.1 protein was expressed in baculovirus-infected cell cultures and in H. virescens larvae. Highly enriched preparations of the secreted VHv1.1 protein were obtained after affinity chromatography using a NTA-(Ni2+) resin. Characterization with purified preparations of the VHv1.1 protein established that the protein is N-glycosylated, containing glycogroups which are PNGase F-sensitive but Endo H-resistant. The recombinant VHv1.1 protein bound to hemocytes in vitro and in vivo and was endocytosed in a manner similar to the native protein produced in CsPDV-infected larvae.
Soldevila
A I
AI
Department of Entomology, University of Kentucky, Lexington 40546, USA.
Heuston
S
S
Webb
B A
BA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002846:Chromatography, Affinity; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006434:Hemocytes / Q000378:metabolism; D006639:Histidine; D009036:Moths; D010455:Peptides / Q000235:genetics*; D018071:Polydnaviridae / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics
D010455:Peptides; D011994:Recombinant Proteins; C099400:VHv1.1 protein, Campoletis sonorensis polydnavirus; D014764:Viral Proteins; C033223:polyhistidine; D006639:Histidine