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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 l.jpg

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 l.jpg

Baculovirus-Insect CellExpression System

  • Binary system.

  • Recombinant baculovirus vector.

    • Delivers gene of interest.

  • Insect cell host.

    • Produces protein product.


Advantages baculovirus insect cell system l.jpg

AdvantagesBaculovirus-Insect Cell System

  • High level gene expression.

    • Strong promoter from viral polh gene.

  • Eucaryotic protein processing.

    • Glycosylation.

Jarvis, 1997


Protein glycosylation l.jpg

Protein Glycosylation

  • Common covalent modification.

  • Can influence protein function.

  • Elaborate biochemical pathways.

  • N-glycosylation.


Mammalian n glycosylation pathway l.jpg

Mammalian N-glycosylationPathway


Mammalian n glycans l.jpg

Mammalian N-glycans


Major insect n glycans l.jpg

Major Insect N-glycans


Major insect vs mammalian n glycans l.jpg

Major Insect vs Mammalian N-Glycans

Marchal et al., 2001


Glycoprotein sialylation l.jpg

Glycoprotein Sialylation

  • Functionally significant.

  • Influences glycoprotein behavior.

    • Nonsialylated gP rapidly cleared in vivo.


Major disadvantage baculovirus insect cell system l.jpg

Major DisadvantageBaculovirus-Insect Cell System

  • Truncated N-glycosylation pathway.

  • Cannot produce sialylated N-glycans.

Marchal et al., 2001


How to address this problem l.jpg

How to Address this Problem?

  • Metabolic engineering.

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


Humanizing insect protein glycosylation pathways l.jpg

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 glycans13 l.jpg

Major Insect vs Mammalian N-Glycans


Immediate early expression plasmids l.jpg

Immediate Early Expression Plasmids


A dual immediate early expression plasmid l.jpg

A Dual Immediate Early Expression Plasmid


Creating transgenic insect cell lines l.jpg

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 l.jpg

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 l.jpg

gp64 Lectin Blots

No competing sugars

1-Sf9

2-Sfß4GalT

3-Sfß4GalT/ST6

Competing sugars

Hollister and Jarvis, 2001


Hpaec pad results l.jpg

HPAEC-PAD Results

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

Hollister et al., 2002


Slide20 l.jpg

MALDI-TOF Results

1079

Sf9

*

*

*

933

1445

Relative Abundance [ % ]

Sfß4GalT

1079

*

*

1079

1445

Sfß4GalT/ST6

1758

1736

1283

1607

Hollister et al., 2002


Conclusions l.jpg

Conclusions

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

    • Galactosylated and sialylated.

  • But, they were monoantennary.

    • Only a3 branch was elongated.


Next question l.jpg

Next Question

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


Major insect vs mammalian n glycans23 l.jpg

Major Insect vs Mammalian N-Glycans


A new transgenic cell line sfswt 1 l.jpg

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 l.jpg

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 results26 l.jpg

HPAEC-PAD Results

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

SfSWT-1

GalGlcNAc2M3F

SialylGalGlcNAc2M3F

Hollister et al., 2002


Maldi tof results l.jpg

Sf9

MALDI-TOF Results

Sfß4GalT

Sfß4GalT/ST6

SfSWT-1

Hollister et al., 2002


Slide28 l.jpg

ESI-MS/MS Results

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


Slide29 l.jpg

ESI-MS/MS Results

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


Conclusions 2 l.jpg

Conclusions 2

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

  • Commercially available.

    • “MIMIC™” (Invitrogen).


Requirements for gp sialylation l.jpg

Requirements for gP sialylation

  • Sialyltransferase.

  • Acceptor substrate (terminally galactosylated).

  • Donor substrate (CMP-sialic acid).

  • CMP-sialic acid transporter.


New questions l.jpg

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 l.jpg

Effects of FBS on Glycosylation by Transgenic Insect Cell Lines

FCS

NO FCS

(Sfß4GalT/ST6)

Hollister et al., 2003


Fbs factor is a sialoglycoprotein l.jpg

SFM + Fetuin

SFM + Asialofetuin

FBS Factor is a Sialoglycoprotein

Hollister et al., 2003


Conclusions 3 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

Lectin Blotting Results

Antibody

Sial-specific lectin

Lectin + competing sugar

Aumiller et al., 2003


Hpaec pad results40 l.jpg

HPAEC-PAD Results

SfSWT-3: SFM

SfSWT-3: SFM/ManNAc

SfSWT-3: SFM/ManNAc

Neuraminidase control

Aumiller et al., 2003


Overall summary l.jpg

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.


Acknowledgements l.jpg

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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