2. 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

3. Baculovirus-Insect Cell Expression System l Binary system. l Recombinant baculovirus vector. l Delivers gene of interest. l Insect cell host. l Produces protein product.

4. Advantages Baculovirus-Insect Cell System l High level gene expression. l Strong promoter from viral polh gene. l Eucaryotic protein processing. l Glycosylation. Jarvis, 1997

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

6. Mammalian N-glycosylation Pathway

7. Mammalian N-glycans

8. Major Insect N-glycans

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

10. Glycoprotein Sialylation l Functionally significant. l Influences glycoprotein behavior. l Nonsialylated gP rapidly cleared in vivo.

11. l Truncated N-glycosylation pathway. l Cannot produce sialylated N-glycans. Major Disadvantage Baculovirus-Insect Cell System Marchal et al., 2001

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

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

14. Major Insect vs Mammalian N-Glycans

15. Immediate Early Expression Plasmids

16. A Dual Immediate Early Expression Plasmid

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

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

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

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

21. 1079 1445 1283 1607 1736 1758 Relative Abundance [ % ] * * 933 * * * Sf9 Sfß4GalT Sfß4GalT/ST6 MALDI-TOF Results Hollister et al., 2002

22. Conclusions l Transgenic insect cell lines produced partially humanized N-glycans. l Galactosylated and sialylated. l But, they were monoantennary. Only  3 branch was elongated.

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

24. Major Insect vs Mammalian N-Glycans

25. A New Transgenic Cell Line: SfSWT-1 l ß1,4-galactosyltransferase.  2,6-sialyltransferase. l N-acetylglucosaminyltransferase II. l N-acetylglucosaminyltransferase I.  2,3-sialyltransferase. Hollister et al., 2002

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

27. HPAEC-PAD Results SialylGalGlcNAcM3F SialylGalGlcNAc 2 M3F GalGlcNAc 2 M3F M3 M3F GalGlcNAcM3F Sf9 Sfß4GalT Sfß4GalT/ST6 SfSWT-1 Hollister et al., 2002

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

29. 1810 2123 1664 16481283 14451607 SfSWT-1 ESI-MS/MS Results Hollister et al., 2002

30. 1810 2123 1664 16481283 14451607 SfSWT-1 ESI-MS/MS Results Hollister et al., 2002

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

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

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

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

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

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

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

38. Newest Transgenic Cell Line: SfSWT-3 l ß1,4-galactosyltransferase.  2,6-sialyltransferase. l N-acetylglucosaminyltransferase II. l N-acetylglucosaminyltransferase I.  2,3-sialyltransferase. l Sialic acid synthase. l CMP-sialic acid synthetase Aumiller et al., 2003

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

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

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

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

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