title: High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.
abstract: Two forms of herpes simplex virus glycoprotein gD were recombined into Autographa californica nuclear polyhedrosis virus (baculovirus) and expressed in infected Spodoptera frugiperda (Sf9) cells. Each protein was truncated at residue 306 of mature gD. One form, gD-1(306t), contains the coding sequence of Patton strain herpes simplex virus type 1 gD; the other, gD-1(QAAt), contains three mutations which eliminate all signals for addition of N-linked oligosaccharides. Prior to recombination, each gene was cloned into the baculovirus transfer vector pVT-Bac, which permits insertion of the gene minus its natural signal peptide in frame with the signal peptide of honeybee melittin. As in the case with many other baculovirus transfer vectors, pVT-Bac also contains the promoter for the baculovirus polyhedrin gene and flanking sequences to permit recombination into the polyhedrin site of baculovirus. Each gD gene was engineered to contain codons for five additional histidine residues following histidine at residue 306, to facilitate purification of the secreted protein on nickel-containing resins. Both forms of gD-1 were abundantly expressed and secreted from infected Sf9 cells, reaching a maximum at 96 h postinfection for gD-1(306t) and 72 h postinfection for gD-1(QAAt). Secretion of the latter protein was less efficient than gD-1(306t), possibly because of the absence of N-linked oligosaccharides from gD-1(QAAt). Purification of the two proteins by a combination of immunoaffinity chromatography, nickel-agarose chromatography, and gel filtration yielded products that were > 99% pure, with excellent recovery. We are able to obtain 20 mg of purified gD-1(306t) and 1 to 5 mg of purified gD-1(QAAt) per liter of infected insect cells grown in suspension. Both proteins reacted with monoclonal antibodies to discontinuous epitopes, indicating that they retain native structure. Use of this system for gD expression makes crystallization trials feasible.
authors.affiliation: DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0400.
authors.forename: W P
authors.initials: WP
authors.lastname: Sisk
authors.forename: J D
authors.initials: JD
authors.lastname: Bradley
authors.forename: R J
authors.initials: RJ
authors.lastname: Leipold
authors.forename: A M
authors.initials: AM
authors.lastname: Stoltzfus
authors.forename: M
authors.initials: M
authors.lastname: Ponce de Leon
authors.forename: M
authors.initials: M
authors.lastname: Hilf
authors.forename: C
authors.initials: C
authors.lastname: Peng
authors.forename: G H
authors.initials: GH
authors.lastname: Cohen
authors.forename: R J
authors.initials: RJ
authors.lastname: Eisenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000942:Antigens, Bacterial / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000942:Antigens, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; C043431:M antigen, Brucella melitensis; D006026:Glycoside Hydrolases