Improved Adenovirus Vector Provides Herpes Simplex Virus Ribonucleotide Reductase R1 and R2 Subunits Very Efficiently


We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15–20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Berkner, K.L. 1988. Development of Adenovirus vector for the expression of heterologous genes. BioTechniques 6: 616–629.

  2. 2

    Berkner, K.L. 1992. Expression of heterologous sequences in adenoviral vectors. Curr. Top. Microbiol. Immunol. 158: 39–66.

  3. 3

    Graham, F.L. and Prevec, L. 1992. Adenovirus-based expression vectors and recombinant vaccines, p. 363–390. In: Vaccines: New Approaches to Immunological Problems. R. W. Ellis (Ed.). Butterworth-Heinemann, Boston.

  4. 4

    Gerard, R.D. and Meidell, R.S. 1993. Adenovirus-mediated gene transfer. Trends Cardiovasc. Med. 5: 171–177.

  5. 5

    Ginsberg, H.S. (Ed.). 1984, The Adenoviruses. Plenum Publishing Corp., NY.

  6. 6

    Tooze, J. 1981. Molecular Biology of Tumor Viruses, Second Edition, Part 2, DNA Tumor Viruses. Cold Spring Harbor Laboaratories, Cold Spring Harbor, NY.

  7. 7

    Gluzman, Y., Reichl, H. and Solnick, D. 1982. Helper-free adenovirus type 5 vectors, p, 187–192. In: Eucaryotic Viral Vectors. Edited by Y. Gluzman (Ed.). Cold Spring Harbor Laboratory, NY.

  8. 8

    Bett, A.J., Prevec, L. and Graham, F. 1993. Packaging capacity and stability of human adenovirus type 5 vectors. J. Virol. 67: 5911–5921.

  9. 9

    Graham, F.L., Smiley, J.R., Russell, W.C. and Nairn, R. 1977. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: 59–72.

  10. 10

    Alkhatib, G. and Briedis, D.J. 1988. High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin using an adenovirus type 5 helper-free vector system. J. Virol. 62: 2718–2727.

  11. 11

    Alkhatib, G., Massie, B. and Briedis, D.J. 1988. Expression of bicistronic measles virus P/CmRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon. J. Virol. 62: 4059–4069.

  12. 12

    Alkatib, G., Richardson, C. and Shen, S.-H. 1990. Intracellular processing, glycosylation, and cell-surface expression of the measles virus fusion protein (F) encoded by a recombinant adenovirus. Virol. 175: 262–270.

  13. 13

    Davidson, D. and Hassell, J.A. 1987. Overproduction of polyoma-virus middle T antigen in mammalian cells through the use of an adenovirus vector. J. Virol. 61: 1226–1239.

  14. 14

    Davis, A.R., Kostek, B., Mason, B.B., Hsiao, C.L., Morin, J., Kheer, S.K. and Hung, P.P. 1985. Expression of hepatitis B surface antigen with a recombinant adenovirus. Proc. Natl. Acad. Sci. USA 82: 7560–7564.

  15. 15

    Huang, A., Jacobi, G., Haj-Amed, Y. and Bacchetti, S. 1988. Expression of the HSV-2 ribonucleotide reductase subunits in adenovirus vectors or stably transformed cells: restoration of enzymatic activity by reassociation of enzyme subunits in the absence of other HSV proteins. Virol. 163: 462–470.

  16. 16

    Jacobs, S.C., Stephenson, J.R. and Wilkinson, G.W.G. 1992. High-level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector protection elicited in a murine model. J. Virol. 66: 20865–2095.

  17. 17

    Johnson, D.C., Goutam, G.-C., Smiley, J.R., Fallis, L. and Graham, F. 1988. Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector. Virol. 164: 1–14.

  18. 18

    Massie, B., Gluzman, Y. and Hassell, J.A. 1986. Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen. Mol. Cell. Biol. 6: 2872–2883.

  19. 19

    Smith, C.L., Hager, G.L., Pike, J.W. and Marx, S.J. 1991. Overexpression of toe human vitamin D3 receptor in mammalian cells using recombinant adenovirus vectors. Mol. Endocrinol. 5: 867–878.

  20. 20

    Zhu, X., Young, C.S.H. and Silverstein, S. 1988. Adenovirus vector expressing functional herpes simplex virus ICPO. J. Virol. 62: 4544–4553.

  21. 21

    Simanis, V. and Lane, D.P. 1985. An immunoaffinity purification procedure for SV40 large T antigen. Virol. 144: 88–100.

  22. 22

    Lamarche, M., Massie, B., Richer, M., Paradis, H. and Langelier, Y. 1990. High level expression in 293 cells of the herpes simplex virus type 2 ribonucleotide reductase subunit 2 using an adenovirus vector. J. Gen. Virol. 71: 1785–1792.

  23. 23

    Alonso-Caplen, F.V., Katze, M.G. and Krug, R.M. 1988. Efficient transcription, not translation, is dependent on adenovirus tripartite leader sequences at late times of infection. J. Virol. 62: 1606–1616.

  24. 24

    Berkner, K.L. and Sharp, P.A. 1985. Effect of the tripartite leader on synthes is of a non-viral protein in an adenovirus 5 recombinant. Nucl. Acids Res. 13: 841–857.

  25. 25

    Berkner, K.L., Schaffhausen, G.S., Roberts, T.M. and Sharp, P.A. 1987. Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant. J. Virol. 61: 1213–1220.

  26. 26

    Mason, B.B., Davis, A.R., Bhat, B.M., Chengalvala, M., Lubeck, M.D., Zandle, G., Kostek, B., Cholodofsky, S., Dheer, S., Molnar-Kimber, K., Mizutani, S. and Hung, P.P. 1990. Adenovirus vaccine vectors expressing hepatitis B surface antigen: importance of regulatory elements in the adenovirus major late intron. Virol. 177: 452–461.

  27. 27

    Mansour, S.L., Grodzicker, T. and Tjian, R. 1986. Downstream sequences affect transcription initiation from the adenovirus major late promoter. Mol. Cell. Biol 6: 2684–2694.

  28. 28

    Leong, K., Lee, W. and Berk, A.J. 1990. High-level transcription from the Adenovirus major late promoter requires downstream binding sites for late-phage specific factors. J. Virol. 64: 51–60.

  29. 29

    Mondésert, G. and Kedinger, C. 1991. Cooperation between upstream and downstream elements of the adenovirus major late promoter for maximal late phase-specific transcription. Nucl. Acids Res. 19: 3221–3228.

  30. 30

    Berk, A.J. 1986. Adenovirus promoters and E1A transactivation. Ann. Rev. Genet. 20: 45–79.

  31. 31

    Grinnell, B.W., Berg, D.T. and Walls, J. 1986. Activation of the adenovirus and BK late promoters: Effects of the BK virus enhancer and transacting viral early proteins. Mol. Cell. Biol. 6: 3596–3605.

  32. 32

    Paradis, H., Gaudreau, P., Massie, B., Lamarche, N., Guilbault, C., Gravel, S. and Langelier, Y. 1991. Affinity purification of active subunit 1 herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity. J. Biol. Chem. 266: 9647–9651.

  33. 33

    Atta, M., Lamarche, N., Battioni, J.P., Massie, B., Langelier, Y., Mansey, D. and Fontecave, M. 1993. Escherichia coli and herpes simplex virus ribonucleotide reductase R2 subunit: compared reactivities of redox centers. Biochem. J. 290: 897–810.

  34. 34

    Lamarche, N., Massie, B., Fontecave, M., Atta, M., Guilbault, C., Dumas, F.P. and Langelier, Y. 1994. Production of herpes simplex virus ribonucleotide reductase R2 subunit with prokaryotic and eukaryotic expression systems: Higher activity of R2 produced by eukaryotic cells related to tyrosyl free radical content. (Submitted).

  35. 35

    Garnier, A., Côté, J., Nadeau, I., Kamen, A. and Massie, B. 1994. Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells. Cytotechnology 15: 145–155.

  36. 36

    O'Reilly, K.R., Miller, L.K. and Luckow, V.A. 1992. Baculovints Expression Vectors, A Laboratory Manual. W. H. Freeman and Company, NY.

  37. 37

    Caron, A.W., Archambault, J. and Massie, B. 1990. High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system. Biotechnol. Bioeng. 36: 1133–1140.

  38. 38

    Caron, A.W., Tom, R.L., Kamen, A.A. and Massie, B. 1994. Baculovirus expression system scaleup by perfusion of high-density Sf-9 cultures. Biotechnol. Bioeng. 43: 881–891.

  39. 39

    Lankinen, H., McLauchlan, J., Weir, M., Furlong, J., Conner, J., McGarrity, A., Mistry, A., Clements, J.B. and Marsden, H.S. 1991. Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli. J. Gen. Virol. 72: 1383–1392.

  40. 40

    Bucham, A.R. and Berg, P. 1988. Comparison of intron-dependent and intron independent gene expression. Mol. Cell. Biol. 8: 4395–1405.

  41. 41

    Zhao, Z., Bouchard, P., Diltz, C.D., Shen, S.-H. and Fischer, E.H. 1993. Purification and characterization of a protein tyrosine phosphatase containing SH2 domains. J. Biol. Chem. 268: 2816–2820.

  42. 42

    Lanford, R.E. 1988. Expression of SV-40 T antigen in insect cells using a baculovirus expression vector. Virol. 167: 72–81.

  43. 43

    Takeuchi-Suzuki, E., Tanaka, T., Hink, W.F. and King, M.M. 1992. High-level expression using baculovirus, purification, and characterization of a monomeric form of type 11 calmodulin-dependent protein kinase. Protein Expression Purif. 3: 160–164.

  44. 44

    Lanford, R.E., Luckow, V., Kennedy, R.C., Dreesman, G.R., Notvall, L. and Summers, M.D. 1989. Expression and characterization of Hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system. J. Virol. 63: 1549–1557.

  45. 45

    Roy, P., Adachie, A., Urakawa, T., Booth, T.F. and Thomas, C.P. 1990. Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells. J. Virol. 64: 1–8.

  46. 46

    Zhang, J., Kalogerakis, N., Behie, L.A. and Latrou, K. 1993. A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system. Biotechnol. Bioeng. 42: 357–366.

  47. 47

    Ellis, R.J. and van der Vies, S. 1991. Molecular chaperones. Ann. Rev. Biochem. 60: 321–347.

  48. 48

    Gething, M.J. and Sambrook, J. 1992. Protein folding in the cell. Nature 355: 33–35.

  49. 49

    Hendrick, J.P. and Hartl, F.U. 1993. Molecular chaperone functions of heat-shock proteins. Ann. Rev. Biochem. 62: 349–384.

  50. 50

    Denis, D., Falgueyret, J.P., Riendeau, D. and Abramovitz, M. 1991. Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors. J. Biol. Chem. 266: 5072–5079.

  51. 51

    Berndt, N. and Cohen, P.T.W. 1990. Renaturation of protein phosphatase expressed at high levels in insect cells using a baculovirus vector. Eur. J. Biochem. 190: 291–297.

  52. 52

    Alnemri, E.S. and Litwack, G. 1993. The steroid binding domain influences intracellular solubility of the baculovirus overexpressed glucocorticoid and mineralocorticoid receptors. Biochemistry 32: 5387–5393.

  53. 53

    Aris, J.P., Basta, P.V., Holmes, W.D., Ballas, L.M., Moomaw, C., Rankl, N.B., Blomel, G., Loomis, C. and Bums, D.J. 1993. Molecular and biochemical characterization of a recombinant human PCK-δ family member. Biochim. Biophys. Acta 1174: 171–181.

  54. 54

    van Drunen Littel-van den Hurk, S., Massie, B., Van den Hurk, J.V., Harland, R., Babiuk, L.A. and Zamb, T.J. 1993. Protection of cattle from BHV-1 infection by immunization with recombinant glycoprotein g1V. Vaccine 11: 25–35.

  55. 55

    Rankl, N.B., Rice, J.W., Gurganus, T.M., Barbee, J.L. and Burns, D.J. 1994. The production of an active protein kinase C-δ in insect cells is greatly enhanced by the use of the basic protein promoter. Protein Expression and Purification 5: 346–356.

  56. 56

    Sridhar, P., Panda, A., Pal, R., Talwar, G.P. and Hasnain, S.E. 1993. Temporal nature of the promoter and not relative strenght determines the expression of an extensively processed protein in a baculovirus system. FEBS Letts. 315: 282–286.

  57. 57

    Summers, M.D. and Smith, G.E. 1987. A Manual of Methods of Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station Research Bulletin no. 1555, Texas agricultural experiment Station, College Sation, TX, 1987.

  58. 58

    Seif, I., Koury, G. and Dhar, R. 1979. The genome of human papovavirus BKV. Cell 18: 963–977.

  59. 59

    Kozak, M. 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. Mol. Biol. 196: 947–950.

  60. 60

    Richardson, C.D., Banville, M., Lalumière, M., Vialard, J. and Meighen, E.A. 1992. Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. Intervirol. 34: 213–227.

  61. 61

    Cohen, E.A., Gaudreau, P., Brazeau, P. and Langelier, Y. 1986. Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2. J. Virol. 60: 1130–1133.

  62. 62

    Vialard, J., Lalumière, M., Vernet, T., Briebis, D., Alkhaib, G., Henning, D., Levin, D. and Richardson, C.D. 1990. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the β-galactosidase gene. J. Virol. 64: 37–50.

  63. 63

    Mann, G.J., Gräslund, A., Ochiai, E.-I., Ingemarson, R. and Thelander, L. 1991 Purification of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry 30: 1939–1947.

  64. 64

    Cohen, E.A., Charron, J., Perret, J. and Langelier, Y. 1985. Herpes simplex virus ribonucleotide reductase induced in infected BHK-21/C13 cells: biochemical evidence for the existence of two non-identical subunits, H1 and H2. J. Gen. Virol. 66: 733–745.

Download references

Author information



Corresponding author

Correspondence to Bernard Massie.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Massie, B., Dionne, J., Lamarche, N. et al. Improved Adenovirus Vector Provides Herpes Simplex Virus Ribonucleotide Reductase R1 and R2 Subunits Very Efficiently. Nat Biotechnol 13, 602–608 (1995).

Download citation

Further reading