Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. 1994 Feb; 68(2): 766–775.
PMCID: PMC236513

High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.


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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Berman PW, Dowbenko D, Lasky LA, Simonsen CC. Detection of antibodies to herpes simplex virus with a continuous cell line expressing cloned glycoprotein D. Science. 1983 Nov 4;222(4623):524–527. [PubMed]
  • Berman PW, Gregory T, Crase D, Lasky LA. Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D. Science. 1985 Mar 22;227(4693):1490–1492. [PubMed]
  • Burke RL. Contemporary approaches to vaccination against herpes simplex virus. Curr Top Microbiol Immunol. 1992;179:137–158. [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Cohen GH, Long D, Matthews JT, May M, Eisenberg R. Glycopeptides of the type-common glycoprotein gD of herpes simplex virus types 1 and 2. J Virol. 1983 Jun;46(3):679–689. [PMC free article] [PubMed]
  • Cohen GH, Dietzschold B, Ponce de Leon M, Long D, Golub E, Varrichio A, Pereira L, Eisenberg RJ. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody. J Virol. 1984 Jan;49(1):102–108. [PMC free article] [PubMed]
  • Cohen GH, Isola VJ, Kuhns J, Berman PW, Eisenberg RJ. Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting. J Virol. 1986 Oct;60(1):157–166. [PMC free article] [PubMed]
  • Cohen GH, Muggeridge MI, Long D, Sodora DA, Eisenberg RJ. Structural and functional studies of herpes simplex virus glycoprotein D. Adv Exp Med Biol. 1992;327:217–228. [PubMed]
  • Doms RW, Lamb RA, Rose JK, Helenius A. Folding and assembly of viral membrane proteins. Virology. 1993 Apr;193(2):545–562. [PubMed]
  • Eisenberg RJ, Long D, Hogue-Angeletti R, Cohen GH. Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2. J Virol. 1984 Jan;49(1):265–268. [PMC free article] [PubMed]
  • Eisenberg RJ, Long D, Pereira L, Hampar B, Zweig M, Cohen GH. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1. J Virol. 1982 Feb;41(2):478–488. [PMC free article] [PubMed]
  • Eisenberg RJ, Long D, Ponce de Leon M, Matthews JT, Spear PG, Gibson MG, Lasky LA, Berman P, Golub E, Cohen GH. Localization of epitopes of herpes simplex virus type 1 glycoprotein D. J Virol. 1985 Feb;53(2):634–644. [PMC free article] [PubMed]
  • Eisenberg RJ, Ponce de Leon M, Friedman HM, Fries LF, Frank MM, Hastings JC, Cohen GH. Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2. Microb Pathog. 1987 Dec;3(6):423–435. [PubMed]
  • Eisenberg RJ, Ponce de Leon M, Pereira L, Long D, Cohen GH. Purification of glycoprotein gD of herpes simplex virus types 1 and 2 by use of monoclonal antibody. J Virol. 1982 Mar;41(3):1099–1104. [PMC free article] [PubMed]
  • Feenstra V, Hodaie M, Johnson DC. Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains. J Virol. 1990 May;64(5):2096–2102. [PMC free article] [PubMed]
  • Fuller AO, Lee WC. Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration. J Virol. 1992 Aug;66(8):5002–5012. [PMC free article] [PubMed]
  • Ghiasi H, Nesburn AB, Kaiwar R, Wechsler SL. Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D. Arch Virol. 1991;121(1-4):163–178. [PubMed]
  • Herold BC, WuDunn D, Soltys N, Spear PG. Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J Virol. 1991 Mar;65(3):1090–1098. [PMC free article] [PubMed]
  • Isola VJ, Eisenberg RJ, Siebert GR, Heilman CJ, Wilcox WC, Cohen GH. Fine mapping of antigenic site II of herpes simplex virus glycoprotein D. J Virol. 1989 May;63(5):2325–2334. [PMC free article] [PubMed]
  • Johnson DC, Burke RL, Gregory T. Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells. J Virol. 1990 Jun;64(6):2569–2576. [PMC free article] [PubMed]
  • Johnson DC, Ligas MW. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J Virol. 1988 Dec;62(12):4605–4612. [PMC free article] [PubMed]
  • Krishna S, Blacklaws BA, Overton HA, Bishop DH, Nash AA. Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: protective responses and T cell recognition of the recombinant-infected cell extracts. J Gen Virol. 1989 Jul;70(Pt 7):1805–1814. [PubMed]
  • Landolfi V, Zarley CD, Abramovitz AS, Figueroa N, Wu SL, Blasiak M, Ishizaka ST, Mishkin EM. Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge. Vaccine. 1993;11(4):407–414. [PubMed]
  • Long D, Wilcox WC, Abrams WR, Cohen GH, Eisenberg RJ. Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2. J Virol. 1992 Nov;66(11):6668–6685. [PMC free article] [PubMed]
  • Miller LK. Baculoviruses as gene expression vectors. Annu Rev Microbiol. 1988;42:177–199. [PubMed]
  • Mishkin EM, Fahey JR, Kino Y, Klein RJ, Abramovitz AS, Mento SJ. Native herpes simplex virus glycoprotein D vaccine: immunogenicity and protection in animal models. Vaccine. 1991 Mar;9(3):147–153. [PubMed]
  • Petrovskis EA, Timmins JG, Armentrout MA, Marchioli CC, Yancey RJ, Jr, Post LE. DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation. J Virol. 1986 Aug;59(2):216–223. [PMC free article] [PubMed]
  • Serafini-Cessi F, Dall'Olio F, Malagolini N, Pereira L, Campadelli-Fiume G. Comparative study on O-linked oligosaccharides of glycoprotein D of herpes simplex virus types 1 and 2. J Gen Virol. 1988 Apr;69(Pt 4):869–877. [PubMed]
  • Sisk WP, Bradley JD, Seivert LL, Vargas RA, Horlick RA. An improved method for rapid screening of baculovirus recombinant plaques by PCR amplification. Biotechniques. 1992 Aug;13(2):186–186. [PubMed]
  • Smith GE, Ju G, Ericson BL, Moschera J, Lahm HW, Chizzonite R, Summers MD. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8404–8408. [PMC free article] [PubMed]
  • Sodora DL, Cohen GH, Eisenberg RJ. Influence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D. J Virol. 1989 Dec;63(12):5184–5193. [PMC free article] [PubMed]
  • Sodora DL, Cohen GH, Muggeridge MI, Eisenberg RJ. Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein. J Virol. 1991 Aug;65(8):4424–4431. [PMC free article] [PubMed]
  • Sodora DL, Eisenberg RJ, Cohen GH. Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides. J Virol. 1991 Aug;65(8):4432–4441. [PMC free article] [PubMed]
  • Tessier DC, Thomas DY, Khouri HE, Laliberté F, Vernet T. Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene. 1991 Feb 15;98(2):177–183. [PubMed]
  • Thomsen DR, Post LE, Elhammer AP. Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9. J Cell Biochem. 1990 May;43(1):67–79. [PubMed]
  • Varghese JN, Laver WG, Colman PM. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature. 1983 May 5;303(5912):35–40. [PubMed]
  • Watson RJ. DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene. Gene. 1983 Dec;26(2-3):307–312. [PubMed]
  • Watson RJ, Weis JH, Salstrom JS, Enquist LW. Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli. Science. 1982 Oct 22;218(4570):381–384. [PubMed]
  • Webster RG, Air GM, Metzger DW, Colman PM, Varghese JN, Baker AT, Laver WG. Antigenic structure and variation in an influenza virus N9 neuraminidase. J Virol. 1987 Sep;61(9):2910–2916. [PMC free article] [PubMed]
  • Whitford M, Faulkner P. A structural polypeptide of the baculovirus Autographa californica nuclear polyhedrosis virus contains O-linked N-acetylglucosamine. J Virol. 1992 Jun;66(6):3324–3329. [PMC free article] [PubMed]
  • Wilcox WC, Long D, Sodora DL, Eisenberg RJ, Cohen GH. The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D. J Virol. 1988 Jun;62(6):1941–1947. [PMC free article] [PubMed]
  • Wiley DC, Wilson IA, Skehel JJ. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981 Jan 29;289(5796):373–378. [PubMed]
  • Wilson IA, Skehel JJ, Wiley DC. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. [PubMed]
  • Zarling JM, Moran PA, Burke RL, Pachl C, Berman PW, Lasky LA. Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD. J Immunol. 1986 Jun 15;136(12):4669–4673. [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...