Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2004 Mar;44(3):93–102. doi: 10.1007/s10616-005-0703-4

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Volker M Gorenflo 1,2,✉, Tom A Pfeifer 3, Gary Lesnicki 1, Emily M Kwan 4, Thomas A Grigliatti 3, Douglas G Kilburn 1,4, James M Piret 1,2
PMCID: PMC3449484  PMID: 19003232

Abstract

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 × 107 cells ml−1 and a final saCBMFX concentration of 4 mg l−1. The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day−1. The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 × 107 Sf9 cells ml−1 were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 107 cells ml−1 the saCBMFX production rate was doubled to 6 mg l−1 day−1. The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l−1 day−1) by an order of magnitude.

Key words: Acoustic filter, Backflush mode, Continuous culture, Insect cells, Protein purification, Serine protease

Full Text

The Full Text of this article is available as a PDF (133.6 KB).

References

  1. Assouline Z., Shen H., Kilburn D.G., Warren R.A. Production and properties of a factor X-cellulose-binding domain fusion protein. Protein Eng. 1993;6:787–792. doi: 10.1093/protein/6.7.787. [DOI] [PubMed] [Google Scholar]
  2. Chisti Y. Hydrodynamic damage to animal cells. Crit. Rev. Biotechnol. 2001;21:67–110. doi: 10.1080/20013891081692. [DOI] [PubMed] [Google Scholar]
  3. Chu L., Robinson D.K. Industrial choices for protein production by large-scale cell culture. Curr. Opin. Biotechnol. 2001;12:180–187. doi: 10.1016/S0958-1669(00)00197-X. [DOI] [PubMed] [Google Scholar]
  4. Cook B.C., Rudolph A.E., Kurumbail R.G., Porche-Sorbet R., Miletich J.P. Directed glycosylation of human coagulation factor X at residue 333. Insight into factor Va-dependent prothrombin catalysis. J. Biol. Chem. 2000;275:38774–38779. doi: 10.1074/jbc.M004573200. [DOI] [PubMed] [Google Scholar]
  5. Scipio R.G., Hermodson M.A., Yates S.G., Davie E.W. A comparison of human prothrombin, factor IX (christmas factor), factor X (stuart factor) and protein S. Biochemistry. 1977;16:698–706. doi: 10.1021/bi00623a022. [DOI] [PubMed] [Google Scholar]
  6. Fann C.H., Guarna M.M., Kilburn D.G., Piret J.M. Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells. Biotechnol. Bioeng. 1999;63:464–472. doi: 10.1002/(SICI)1097-0290(19990520)63:4<464::AID-BIT10>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  7. Garber K. Biotech industry faces new bottleneck. Nat. Biotechnol. 2001;19:184–185. doi: 10.1038/85559. [DOI] [PubMed] [Google Scholar]
  8. Gorenflo V.M., Angepat S., Bowen B.D., Piret J.M. Optimization of an acoustic cell filter with a novel air-backflush system. Biotechnol. Prog. 2003;19:30–36. doi: 10.1021/bp025625a. [DOI] [PubMed] [Google Scholar]
  9. Guarna M.M., Cote H.C., Kwan E.M., Rintoul G.L., Meyhack B., Heim J., MacGillivray R.T., Warren R.A., Kilburn D.G. Factor X fusion proteins: Improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein. Protein Expr. Purif. 2000;20:133–141. doi: 10.1006/prep.2000.1292. [DOI] [PubMed] [Google Scholar]
  10. Hegedus D.D., Pfeifer T.A., Hendry J., Theilmann D.A., Grigliatti T.A. A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines. Gene. 1998;207:241–249. doi: 10.1016/S0378-1119(97)00636-7. [DOI] [PubMed] [Google Scholar]
  11. Hegedus D.D., Pfeifer T.A., Theilmann D.A., Kennard M.L., Gabathuler R., Jefferies W.A., Grigliatti T.A. Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines. Protein Expr. Purif. 1999;15:296–307. doi: 10.1006/prep.1998.1021. [DOI] [PubMed] [Google Scholar]
  12. Himmelspach M., Pfleiderer M., Fischer B.E., Plaimauer B., Antoine G., Falkner F.G., Dorner F., Schlokat U. Recombinant human factor X: High yield expression and the role of furin in proteolytic maturation in vivo and in vitro. Thromb. Res. 2000;97:51–67. doi: 10.1016/S0049-3848(99)00145-0. [DOI] [PubMed] [Google Scholar]
  13. Inoue K., Morita T. Identification of o-linked oligosaccharide chains in the activation peptides of blood coagulation factor X. The role of the carbohydrate moieties in the activation of factor X. Eur. J. Biochem. 1993;218:153–163. doi: 10.1111/j.1432-1033.1993.tb18361.x. [DOI] [PubMed] [Google Scholar]
  14. Keane J.T., Ryan D., Gray P.P. Effect of shear stress on expression of a recombinant protein by chinese hamster ovary cells. Biotechnol. Bioeng. 2003;81:211–220. doi: 10.1002/bit.10472. [DOI] [PubMed] [Google Scholar]
  15. Kunas K.T., Papoutsakis E.T. The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors. J. Biotechnol. 1990;15:57–69. doi: 10.1016/0168-1656(90)90051-C. [DOI] [PubMed] [Google Scholar]
  16. Kwan E., Guarna M.M., Boraston A.B., Gilkes N.R., Haynes C.A., Kilburn D.G., Warren R.A. Self-activating factor X derivative fused to the C-terminus of a cellulose-binding module: Production and properties. Biotechnol. Bioeng. 2002;79:724–732. doi: 10.1002/bit.10312. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage t4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Leytus S.P., Chung D.W., Kisiel W., Kurachi K., Davie E.W. Characterization of a cDNA coding for human factor X. Proc. Natl Acad. Sci. USA. 1984;81:3699–3702. doi: 10.1073/pnas.81.12.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ma N., Koelling K.W., Chalmers J.J. Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces. Biotechnol. Bioeng. 2002;80:428–437. doi: 10.1002/bit.10387. [DOI] [PubMed] [Google Scholar]
  20. McQueen A., Bailey J.E. Influence of serum level, cell-line, flow type and viscosity on flow-induced lysis of suspended mammalian-cells. Biotechnol. Lett. 1989;11:531–536. doi: 10.1007/BF01040030. [DOI] [Google Scholar]
  21. Merten O.-W. Constructive improvement of the ultrasonic separation device ADI 1015. Cytotechnology. 2000;34:175–179. doi: 10.1023/A:1008147822625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Messier T.L., Pittman D.D., Long G.L., Kaufman R.J., Church W.R. Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X. Gene. 1991;99:291–294. doi: 10.1016/0378-1119(91)90141-W. [DOI] [PubMed] [Google Scholar]
  23. Pfeifer T.A., Hegedus D.D., Grigliatti T.A., Theilmann D.A. Baculovirus immediate-early promoter-mediated expression of the zeocin resistance gene for use as a dominant selectable marker in dipteran and lepidopteran insect cell lines. Gene. 1997;188:183–190. doi: 10.1016/S0378-1119(96)00756-1. [DOI] [PubMed] [Google Scholar]
  24. Pfeifer T.A., Guarna M.M., Kwan E.M., Lesnicki G., Theilmann D.A., Grigliatti T.A., Kilburn D.G. Expression analysis of a modified factor X in stably transformed insect cell lines. Protein Expr. Purif. 2001;23:233–241. doi: 10.1006/prep.2001.1503. [DOI] [PubMed] [Google Scholar]
  25. Rezaie A.R., Neuenschwander P.F., Morrissey J.H., Esmon C.T. Analysis of the functions of the first epidermal growth factor-like domain of factor X. J. Biol. Chem. 1993;268:8176–8180. [PubMed] [Google Scholar]
  26. Rudolph A.E., Mullane M.P., Porche-Sorbet R., ‘Miletich J.P. Expression, purification and characterization of recombinant human factor X. Protein Expr. Purif. 1997;10:373–378. doi: 10.1006/prep.1997.0752. [DOI] [PubMed] [Google Scholar]
  27. Stanton C., Wallin R. Processing and trafficking of clotting factor X in the secretory pathway. Effects of warfarin. Biochem. J. 1992;284:25–31. doi: 10.1042/bj2840025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stanton C., Ross R.P., Hutson S., Wallin R. Processing and expression of rat and human clotting factor-X-encoding cDNAs. Gene. 1996;169:269–273. doi: 10.1016/0378-1119(95)00832-2. [DOI] [PubMed] [Google Scholar]
  29. Trampler F., Sonderhoff S.A., Pui P.W., Kilburn D.G., Piret J.M. Acoustic cell filter for high density perfusion culture of hybridoma cells. Bio/Technology (N Y) 1994;12:281–284. doi: 10.1038/nbt0394-281. [DOI] [PubMed] [Google Scholar]
  30. Wallin R., Martin L.F. Early processing of prothrombin and factor X by the vitamin K-dependent carboxylase. J. Biol. Chem. 1988;263:9994–10001. [PubMed] [Google Scholar]
  31. Zhang J., Collins A., Chen M., Knyazev I., Gentz R. High-density perfusion culture of insect cells with a biosep ultrasonic filter. Biotechnol. Bioeng. 1998;59:351–359. doi: 10.1002/(SICI)1097-0290(19980805)59:3<351::AID-BIT11>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES