Producing Recombinant Glycoproteins in the Baculovirus-Insect Cell System

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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

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Baculovirus-Insect CellExpression System

• Binary system.
• Recombinant baculovirus vector.
• Delivers gene of interest.
• Insect cell host.
• Produces protein product.

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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.

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Mammalian N-glycosylationPathway
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Mammalian N-glycans
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Major Insect N-glycans
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Major Insect vs Mammalian N-Glycans
Marchal et al., 2001
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Glycoprotein Sialylation

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

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Major DisadvantageBaculovirus-Insect Cell System

• Truncated N-glycosylation pathway.
• Cannot produce sialylated N-glycans.

Marchal et al., 2001
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How to Address this Problem?

• Metabolic engineering.
• Genetically modify (humanize) insect protein
  N-glycosylation pathway.

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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
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Major Insect vs Mammalian N-Glycans
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Immediate Early Expression Plasmids
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A Dual Immediate Early Expression Plasmid
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Creating Transgenic Insect Cell Lines

• Constructed pIE1ß4GalT, pIE1ST6, pIE1Neo.
• Cotransfected Sf9 or High Five cells.
• Isolated drug-resistant clones.
• Screened for glycosyltransferase expression.

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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
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gp64 Lectin Blots
No competing sugars
1-Sf9 2-Sfß4GalT 3-Sfß4GalT/ST6
Competing sugars
Hollister and Jarvis, 2001
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HPAEC-PAD Results
M3F
Sf9
M3
Sfß4GalT
GalGlcNAcM3F
Sfß4GalT/ST6
SialylGalGlcNAcM3F
Hollister et al., 2002
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MALDI-TOF Results
1079
Sf9



933
1445
Relative Abundance
Sfß4GalT
1079


1079
1445
Sfß4GalT/ST6
1758
1736
1283
1607
Hollister et al., 2002
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Conclusions

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

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Next Question

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

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Major Insect vs Mammalian N-Glycans
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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
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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
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HPAEC-PAD Results
M3F
Sf9
M3
Sfß4GalT
GalGlcNAcM3F
Sfß4GalT/ST6
SialylGalGlcNAcM3F
SfSWT-1
GalGlcNAc2M3F
SialylGalGlcNAc2M3F
Hollister et al., 2002
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MALDI-TOF Results
Sf9
Sfß4GalT
Sfß4GalT/ST6
SfSWT-1
Hollister et al., 2002
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ESI-MS/MS Results
SfSWT-1
1810
2123
1648
1283
1664
1445
1607
Hollister et al., 2002
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ESI-MS/MS Results
SfSWT-1
1810
2123
1648
1283
1664
1445
1607
Hollister et al., 2002
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Conclusions 2

• Sf9 cells were engineered to produce biantennary,
  monosialylated N-glycans.
• Commercially available.
• MIMIC (Invitrogen).

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Requirements for gP sialylation

• Sialyltransferase.
• Acceptor substrate (terminally galactosylated).
• Donor substrate (CMP-sialic acid).
• CMP-sialic acid transporter.

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New Questions

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

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Effects of FBS on Glycosylation by Transgenic
Insect Cell Lines
FCS
NO FCS
(Sfß4GalT/ST6)
Hollister et al., 2003
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FBS Factor is a Sialoglycoprotein
Hollister et al., 2003
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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
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Final Question

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

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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
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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
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Lectin Blotting Results
Antibody
Sial-specific lectin
Lectin competing sugar
Aumiller et al., 2003
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HPAEC-PAD Results
SfSWT-3 SFM
SfSWT-3 SFM/ManNAc
SfSWT-3 SFM/ManNAc Neuraminidase control
Aumiller et al., 2003
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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.

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Acknowledgements

• 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