, Volume 17, Issue 1, pp 21–26 | Cite as

Strong buffering capacity of insect cells. Implications for the baculovirus expression system

  • Miguel Medina
  • Abelardo López-Rivas
  • Douwe Zuidema
  • Graham J. Belsham
  • Esteban Domingo
  • Just M. Vlak


Insect cells are widely used for expression of a variety of different proteins by using the baculovirus expression system. The applicability of this system depends on production of proteins which have biological properties similar to their native counterparts. One application has been the expression of viral capsid proteins and their assembly into empty capsid structures to provide new viral immunogens which retain complex antigenic sites. An important parameter for efficient folding and assembly of proteins into viral procapsids may be the intracellular pH, particularly for acid-labile particles such as foot-and-mouth disease virus (FMDV). Benzoic acid was used as an effective indicator of intracellular pH in insect cells and 3-O-methyl glucose to measure cell volumes. We have determined the intracellular volume of theSpodoptera frugiperda IPLB-Sf21 insect cells 0.50±0.08 pL per cell. Using the distribution of [14C]-benzoic acid, we show that the intracellular pH remains constant at pH 7.0 when the cells are grown in media with pH values ranging from 6.2 to 6.8 and, moreover, is not affected by baculovirus infection. These results suggest that insect cells are suitable to express and produce acid-labile structures via the baculovirus expression system and that assembly of proteins and viral procapsids could occur.

Key words

Insect cells intracellular pH baculovirus foot-and-mouth disease virus empty capsids 


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  1. Acharya R, Fry E, Stuart DI, Fox G, Rowlands D and Brown F (1989) The three-dimensional structure of foot and mouth disease virus at 2.9 A. Nature 337: 709–716.Google Scholar
  2. Barret JT (1983) An introduction to immunochemistry and immunobiology. In: Harshberger SE (ed.) Immunology (pp. 84). Mosby Company, St. Louis.Google Scholar
  3. Brown CS, van Lent JWM, Vlak JM and Spaan WJM (1991) Assembly empty capsids by using baculovirus recombinants expressing human parvovirus B 19 structural proteins. J. Virol. 65: 2702–2706.Google Scholar
  4. Doppler W, Jaggi R and Groner B (1987) Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression. Gene 54: 147–153.Google Scholar
  5. French TJ, Marshall JJA and Roy P (1990) Assembly of double-shelled, viruslike particles of bluetongue virus using simultaneous expression of four structural proteins. J. Virol. 64: 5695–5700.Google Scholar
  6. Hink F (1970) Established insect cell line from the cabbage looper Trichoplusia ni. Nature 226: 466–467.Google Scholar
  7. Kletzien RF, Pariza MW, Becker JE and Potter VR (1975) A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture. Anal. Biochem. 68: 537–544.Google Scholar
  8. Kurtz I and Golchini K (1987) Na+-independent Cl−HCO3 exchanges in Madin-Darby canine kidney cells. Role in intracellular pH regulation. J. Biol. Chem. 262: 4516–4220.Google Scholar
  9. L'Allemain G, Paris S and Poysségur J (1984) Growth factor action and intracellular pH regulation in fibroblasts. J. Biol. Chem. 259: 5809–5815.Google Scholar
  10. López-Rivas A, Stroobant P, Waterfield MD and Rozengurt E (1984) Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells. EMBO J. 3: 939–944.Google Scholar
  11. Martínez C, Dalsgaard K, López-de-Turiso JA, Cortés E, Vela C and Casal JI (1992) Production of porcine parvovirus empty capsids with high immunogenic activity. Vaccine 10: 684–690.Google Scholar
  12. Perona R, Portillo F, Giráldez F and Serrano R (1990) Transformation and pH homeostasis of fibroblasts expressing yeast H+-ATPase containing site-directed mutations. Mol. Cell. Biol. 10: 4110–4115.Google Scholar
  13. Roos A and Boron WF (1981) Intracellular pH. Physiol. Rev. 61: 296–434.Google Scholar
  14. Roosien J, Belsham GJ, Ryan MD, King AMQ and Vlak JM (1990) Synthesis of foot-and-mouth disease virus capsid protein in insect cells using baculovirus expression vectors. J. Gen. Virol. 71: 1703–1711.Google Scholar
  15. Rose RC, Bonnez W, Reichman RC and Garcea RL (1993) Expression of human papillomavirus 11 L1 protein in insect cells:in vivo andin vitro assembly of viruslike particles. J. Virol. 67: 1936–1944.Google Scholar
  16. Rueckert RR (1990) Picornaviridae and their replication. In: Fields BN, Knipe DM, Chanock RM, Hirsch MS, Melnick JL, Monath TP and Roizman B (eds.) Virology, 2nd ed. (pp. 507–548). Raven Press, New York.Google Scholar
  17. Sabara M, Parker M, Aha P, Cosco C, Gibbons E, Parasons S and Babiuk LA (1991) Assembly of double-shelled rotaviruslike particles by simultaneous expression of recombinant VP6 and VP7 proteins. J. Virol. 65: 6994–6997.Google Scholar
  18. Saliki JT, Mizak B, Flore HP, Gettig RR, Burand JP, Carmichael LE, Wood HA and Parrish CR (1992) Canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs. J. Gen. Virol. 73: 369–374.Google Scholar
  19. Schuldiner S and Rozengurt E (1982) Na+/H+ antiporter in Swiss 3T3 cells: mitogenic stimulatior leads to cytoplasmic alkalinisation. Proceedings of the National Academy of Sciences USA 79: 7778–7782.Google Scholar
  20. Smith GE and Summers MD (1978) Analysis of baculovirus genomes with restriction endonucleases. Virology 58: 893–899.Google Scholar
  21. Summers MD and Smith GE (1987) A manual of methods for baculovirus vectors and insect cellculture procedures. Texas Agricultural Experimental Station Bulletin 1555.Google Scholar
  22. Takehara K, Ireland D and Bishop DHL (1988) Coexpression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J. Gen. Virol 69: 2763–2777.Google Scholar
  23. Thomsen DR, Meyer AL and Post EL (1992) Expression of feline leukemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression system. J. Gen. Virol. 73: 1819–1824.Google Scholar
  24. Thomsen DR, Roof LL and Homa FL (1994) Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins. J. Virol. 68: 2442–2457.Google Scholar
  25. Urakawa T, Ferguson M, Minor PD, Cooper J, Sullivan M, Almond JW and Bishop DHL (1989) Synthesis of immunogenic, but non-infectious, poliovirus particles in insect cells by a baculovirus expression vector. J. Gen. Virol. 70: 1453–1463.Google Scholar
  26. Vaughn JL, Goodwin RH, Tompkins GJ and McCawley P (1977) The stablishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13: 213–217.Google Scholar
  27. Villalba M, Martínez-Serrano A, Börner C, Blanco P and Satrústegui J (1992) NMDA-induced increase in [Ca++]i and45Ca++ uptake in acutely dissociated brain cells derived from adult rat. Brain Res. 570: 347–353.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Miguel Medina
    • 1
  • Abelardo López-Rivas
    • 2
  • Douwe Zuidema
    • 3
  • Graham J. Belsham
    • 4
  • Esteban Domingo
    • 1
  • Just M. Vlak
    • 3
  1. 1.Centro de Biología Molecular ‘Severo Ochoa’CSICMadridSpain
  2. 2.Institute de Parasitología López-NeyraCSICGranadaSpain
  3. 3.Department of VirologyAgricultural UniversityWageningenThe Netherlands
  4. 4.Pirbright LaboratoryAFRC Institute for Animal HealthPirbrightUK

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