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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 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 Cell Baculovirus-Insect Cell SystemExpression System

  • Binary system.

  • Recombinant baculovirus vector.

    • Delivers gene of interest.

  • Insect cell host.

    • Produces protein product.


Advantages baculovirus insect cell system l.jpg
Advantages Baculovirus-Insect Cell SystemBaculovirus-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 Baculovirus-Insect Cell System

  • Common covalent modification.

  • Can influence protein function.

  • Elaborate biochemical pathways.

  • N-glycosylation.


Mammalian n glycosylation pathway l.jpg
Mammalian Baculovirus-Insect Cell SystemN-glycosylationPathway


Mammalian n glycans l.jpg
Mammalian N-glycans Baculovirus-Insect Cell System


Major insect n glycans l.jpg
Major Insect N-glycans Baculovirus-Insect Cell System


Major insect vs mammalian n glycans l.jpg
Major Insect vs Mammalian N-Glycans Baculovirus-Insect Cell System

Marchal et al., 2001


Glycoprotein sialylation l.jpg
Glycoprotein Sialylation Baculovirus-Insect Cell System

  • Functionally significant.

  • Influences glycoprotein behavior.

    • Nonsialylated gP rapidly cleared in vivo.


Major disadvantage baculovirus insect cell system l.jpg
Major Disadvantage Baculovirus-Insect Cell SystemBaculovirus-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? Baculovirus-Insect Cell System

  • Metabolic engineering.

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


Humanizing insect protein glycosylation pathways l.jpg
Humanizing Insect Protein Glycosylation Pathways Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System


Immediate early expression plasmids l.jpg
Immediate Early Expression Plasmids Baculovirus-Insect Cell System


A dual immediate early expression plasmid l.jpg
A Dual Immediate Early Expression Plasmid Baculovirus-Insect Cell System


Creating transgenic insect cell lines l.jpg
Creating Transgenic Insect Cell Lines Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System

No competing sugars

1-Sf9

2-Sfß4GalT

3-Sfß4GalT/ST6

Competing sugars

Hollister and Jarvis, 2001


Hpaec pad results l.jpg
HPAEC-PAD Results Baculovirus-Insect Cell System

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

Hollister et al., 2002


Slide20 l.jpg

MALDI-TOF Results Baculovirus-Insect Cell System

1079

Sf9

*

*

*

933

1445

Relative Abundance [ % ]

Sfß4GalT

1079

*

*

1079

1445

Sfß4GalT/ST6

1758

1736

1283

1607

Hollister et al., 2002


Conclusions l.jpg
Conclusions Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System


A new transgenic cell line sfswt 1 l.jpg
A New Transgenic Cell Line: Baculovirus-Insect Cell SystemSfSWT-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 Baculovirus-Insect Cell System

  • 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 Baculovirus-Insect Cell System

M3F

Sf9

M3

Sfß4GalT

GalGlcNAcM3F

Sfß4GalT/ST6

SialylGalGlcNAcM3F

SfSWT-1

GalGlcNAc2M3F

SialylGalGlcNAc2M3F

Hollister et al., 2002


Maldi tof results l.jpg

Sf9 Baculovirus-Insect Cell System

MALDI-TOF Results

Sfß4GalT

Sfß4GalT/ST6

SfSWT-1

Hollister et al., 2002


Slide28 l.jpg

ESI-MS/MS Results Baculovirus-Insect Cell System

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


Slide29 l.jpg

ESI-MS/MS Results Baculovirus-Insect Cell System

SfSWT-1

1810

2123

1283

1648

1445

1607

1664

Hollister et al., 2002


Conclusions 2 l.jpg
Conclusions 2 Baculovirus-Insect Cell System

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

  • Commercially available.

    • “MIMIC™” (Invitrogen).


Requirements for gp sialylation l.jpg
Requirements for gP sialylation Baculovirus-Insect Cell System

  • Sialyltransferase.

  • Acceptor substrate (terminally galactosylated).

  • Donor substrate (CMP-sialic acid).

  • CMP-sialic acid transporter.


New questions l.jpg
New Questions Baculovirus-Insect Cell System

  • 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 Lines

SFM + Asialofetuin

FBS Factor is a Sialoglycoprotein

Hollister et al., 2003


Conclusions 3 l.jpg
Conclusions 3 Lines

  • 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 Lines

  • 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: LinesSfSWT-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 Lines

  • 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 Lines

Antibody

Sial-specific lectin

Lectin + competing sugar

Aumiller et al., 2003


Hpaec pad results40 l.jpg
HPAEC-PAD Results Lines

SfSWT-3: SFM

SfSWT-3: SFM/ManNAc

SfSWT-3: SFM/ManNAc

Neuraminidase control

Aumiller et al., 2003


Overall summary l.jpg
Overall Summary Lines

  • 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 Lines

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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