Producing Recombinant Glycoproteins in the Baculovirus-Insect Cell System - PowerPoint PPT Presentation

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Producing Recombinant Glycoproteins in the Baculovirus-Insect Cell System
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Producing Recombinant Glycoproteins in the Baculovirus-Insect Cell System

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  1. Producing Recombinant Glycoproteins in the Baculovirus-Insect Cell System Donald L. Jarvis, Jason R. Hollister, Jared J. Aumiller Department of Molecular Biology University of Wyoming Laramie, WY, USA

  2. Baculovirus-Insect CellExpression System • Binary system. • Recombinant baculovirus vector. • Delivers gene of interest. • Insect cell host. • Produces protein product.

  3. AdvantagesBaculovirus-Insect Cell System • High level gene expression. • Strong promoter from viral polh gene. • Eucaryotic protein processing. • Glycosylation. Jarvis, 1997

  4. Protein Glycosylation • Common covalent modification. • Can influence protein function. • Elaborate biochemical pathways. • N-glycosylation.

  5. Mammalian N-glycosylationPathway

  6. Mammalian N-glycans

  7. Major Insect N-glycans

  8. Major Insect vs Mammalian N-Glycans Marchal et al., 2001

  9. Glycoprotein Sialylation • Functionally significant. • Influences glycoprotein behavior. • Nonsialylated gP rapidly cleared in vivo.

  10. Major DisadvantageBaculovirus-Insect Cell System • Truncated N-glycosylation pathway. • Cannot produce sialylated N-glycans. Marchal et al., 2001

  11. How to Address this Problem? • Metabolic engineering. • Genetically modify (“humanize”) insect protein N-glycosylation pathway.

  12. Humanizing Insect Protein Glycosylation Pathways • Identify missing functions. • Identify human/mammalian genes. • Place under control of insect promoters. • Genetically transform insect cell lines. • Isolate transgenic insect cells that constitutively express these genes. Jarvis et al., 1998; Jarvis et al., 2003, Jarvis, 2003

  13. Major Insect vs Mammalian N-Glycans

  14. Immediate Early Expression Plasmids

  15. A Dual Immediate Early Expression Plasmid

  16. Creating Transgenic Insect Cell Lines • Constructed pIE1ß4GalT, pIE1ST6, pIE1Neo. • Cotransfected Sf9 or High Five™ cells. • Isolated drug-resistant clones. • Screened for glycosyltransferase expression.

  17. Transgenic Insect Cell Lines • Normal morphologies. • Normal growth properties. • Support baculovirus infection. • Support baculovirus gene expression. • Constitutive Gal-T and Sial-T activities. • Can they produce humanized glycoproteins? Breitbach and Jarvis, 2001; Hollister et al., 1998; Hollister and Jarvis 2001

  18. gp64 Lectin Blots No competing sugars 1-Sf9 2-Sfß4GalT 3-Sfß4GalT/ST6 Competing sugars Hollister and Jarvis, 2001

  19. HPAEC-PAD Results M3F Sf9 M3 Sfß4GalT GalGlcNAcM3F Sfß4GalT/ST6 SialylGalGlcNAcM3F Hollister et al., 2002

  20. MALDI-TOF Results 1079 Sf9 * * * 933 1445 Relative Abundance [ % ] Sfß4GalT 1079 * * 1079 1445 Sfß4GalT/ST6 1758 1736 1283 1607 Hollister et al., 2002

  21. Conclusions • Transgenic insect cell lines produced partially humanized N-glycans. • Galactosylated and sialylated. • But, they were monoantennary. • Only a3 branch was elongated.

  22. Next Question • Can insect cells be further humanized to produce BIantennary, sialylated N-glycans?

  23. Major Insect vs Mammalian N-Glycans

  24. A New Transgenic Cell Line: SfSWT-1 • ß1,4-galactosyltransferase. • a2,6-sialyltransferase. • N-acetylglucosaminyltransferase II. • N-acetylglucosaminyltransferase I. • a2,3-sialyltransferase. Hollister et al., 2002

  25. SfSWT-1 Cells • Normal morphology and growth. • Support baculovirus infection. • Support baculovirus gene expression. • Express all five transferase genes. • Can they produce biantennary N-glycans? Hollister et al., 2002

  26. HPAEC-PAD Results M3F Sf9 M3 Sfß4GalT GalGlcNAcM3F Sfß4GalT/ST6 SialylGalGlcNAcM3F SfSWT-1 GalGlcNAc2M3F SialylGalGlcNAc2M3F Hollister et al., 2002

  27. Sf9 MALDI-TOF Results Sfß4GalT Sfß4GalT/ST6 SfSWT-1 Hollister et al., 2002

  28. ESI-MS/MS Results SfSWT-1 1810 2123 1283 1648 1445 1607 1664 Hollister et al., 2002

  29. ESI-MS/MS Results SfSWT-1 1810 2123 1283 1648 1445 1607 1664 Hollister et al., 2002

  30. Conclusions 2 • Sf9 cells were engineered to produce biantennary, monosialylated N-glycans. • Commercially available. • “MIMIC™” (Invitrogen).

  31. Requirements for gP sialylation • Sialyltransferase. • Acceptor substrate (terminally galactosylated). • Donor substrate (CMP-sialic acid). • CMP-sialic acid transporter.

  32. New Questions • Where does the donor CMP-SA come from? • How is it transported into the Golgi?

  33. Effects of FBS on Glycosylation by Transgenic Insect Cell Lines FCS NO FCS (Sfß4GalT/ST6) Hollister et al., 2003

  34. SFM + Fetuin SFM + Asialofetuin FBS Factor is a Sialoglycoprotein Hollister et al., 2003

  35. Conclusions 3 • Sfß4GalT/ST6 and SfSWT-1 cells require FBS or serum sialoglycoproteins for de novo glycoprotein sialylation. • These cells can salvage sialic acids from extracellular serum sialoglycoproteins. Hollister et al., 2003

  36. Final Question • Can we create a transgenic insect cell line that produces humanized recombinant glycoproteins when cultured in SFM?

  37. Newest Transgenic Cell Line: SfSWT-3 • ß1,4-galactosyltransferase. • a2,6-sialyltransferase. • N-acetylglucosaminyltransferase II. • N-acetylglucosaminyltransferase I. • a2,3-sialyltransferase. • Sialic acid synthase. • CMP-sialic acid synthetase Aumiller et al., 2003

  38. SfSWT-3 Cells • Normal morphology and growth. • Support baculovirus infection. • Support baculovirus gene expression. • Express all seven mammalian genes. • Can they produce biantennary, sialylated N-glycans in the absence of serum? Aumiller et al., 2003

  39. Lectin Blotting Results Antibody Sial-specific lectin Lectin + competing sugar Aumiller et al., 2003

  40. HPAEC-PAD Results SfSWT-3: SFM SfSWT-3: SFM/ManNAc SfSWT-3: SFM/ManNAc Neuraminidase control Aumiller et al., 2003

  41. Overall Summary • Genetic engineering can be used to extend insect cell protein glycosylation pathways. • New baculovirus-insect cell systems can produce structurally authentic glycoproteins. • Products appear to be quite homogeneous. • Amenable to crystallization and structural analysis.

  42. Jason Hollister Eric Finn Carla Weinkauf Neung-Seon Seo Jared Aumiller Dale Howe Kevin Breitbach NIH GM49734 Harald Conradt Eckard Grabenhorst Manfred Nimtz Joel Shaper Jim Paulson Harry Schachter Pamela Stanley Shuichi Tsuji Acknowledgements