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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. Baculovirus-Insect Cell Expression System. Binary system.

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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

Donald L. Jarvis, Jason R. Hollister, Jared J. Aumiller

Department of Molecular Biology

University of Wyoming

Laramie, WY, USA


Baculovirus-Insect CellExpression System

  • Binary system.

  • Recombinant baculovirus vector.

    • Delivers gene of interest.

  • Insect cell host.

    • Produces protein product.


AdvantagesBaculovirus-Insect Cell System

  • High level gene expression.

    • Strong promoter from viral polh gene.

  • Eucaryotic protein processing.

    • Glycosylation.

Jarvis, 1997


Protein Glycosylation

  • Common covalent modification.

  • Can influence protein function.

  • Elaborate biochemical pathways.

  • N-glycosylation.


Mammalian N-glycosylationPathway


Mammalian N-glycans


Major Insect N-glycans


Major Insect vs Mammalian N-Glycans

Marchal et al., 2001


Glycoprotein Sialylation

  • Functionally significant.

  • Influences glycoprotein behavior.

    • Nonsialylated gP rapidly cleared in vivo.


Major DisadvantageBaculovirus-Insect Cell System

  • Truncated N-glycosylation pathway.

  • Cannot produce sialylated N-glycans.

Marchal et al., 2001


How to Address this Problem?

  • Metabolic engineering.

  • Genetically modify (“humanize”) insect protein N-glycosylation pathway.


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


Major Insect vs Mammalian N-Glycans


Immediate Early Expression Plasmids


A Dual Immediate Early Expression Plasmid


Creating Transgenic Insect Cell Lines

  • Constructed pIE1ß4GalT, pIE1ST6, pIE1Neo.

  • Cotransfected Sf9 or High Five™ cells.

  • Isolated drug-resistant clones.

  • Screened for glycosyltransferase expression.


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


gp64 Lectin Blots

No competing sugars

1-Sf9

2-Sfß4GalT

3-Sfß4GalT/ST6

Competing sugars

Hollister and Jarvis, 2001


HPAEC-PAD Results

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

Hollister et al., 2002


MALDI-TOF Results

1079

Sf9

*

*

*

933

1445

Relative Abundance [ % ]

Sfß4GalT

1079

*

*

1079

1445

Sfß4GalT/ST6

1758

1736

1283

1607

Hollister et al., 2002


Conclusions

  • Transgenic insect cell lines produced partially humanized N-glycans.

    • Galactosylated and sialylated.

  • But, they were monoantennary.

    • Only a3 branch was elongated.


Next Question

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


Major Insect vs Mammalian N-Glycans


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


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


HPAEC-PAD Results

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

SfSWT-1

GalGlcNAc2M3F

SialylGalGlcNAc2M3F

Hollister et al., 2002


Sf9

MALDI-TOF Results

Sfß4GalT

Sfß4GalT/ST6

SfSWT-1

Hollister et al., 2002


ESI-MS/MS Results

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


ESI-MS/MS Results

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


Conclusions 2

  • Sf9 cells were engineered to produce biantennary, monosialylated N-glycans.

  • Commercially available.

    • “MIMIC™” (Invitrogen).


Requirements for gP sialylation

  • Sialyltransferase.

  • Acceptor substrate (terminally galactosylated).

  • Donor substrate (CMP-sialic acid).

  • CMP-sialic acid transporter.


New Questions

  • Where does the donor CMP-SA come from?

  • How is it transported into the Golgi?


Effects of FBS on Glycosylation by Transgenic Insect Cell Lines

FCS

NO FCS

(Sfß4GalT/ST6)

Hollister et al., 2003


SFM + Fetuin

SFM + Asialofetuin

FBS Factor is a Sialoglycoprotein

Hollister et al., 2003


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


Final Question

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


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


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


Lectin Blotting Results

Antibody

Sial-specific lectin

Lectin + competing sugar

Aumiller et al., 2003


HPAEC-PAD Results

SfSWT-3: SFM

SfSWT-3: SFM/ManNAc

SfSWT-3: SFM/ManNAc

Neuraminidase control

Aumiller et al., 2003


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.


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


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