Production and Glycosylation Analysis of Model Proteins from a Vaccinia Virus-Mammalian Cell Expression System

Nicole A. Bleckwenna,b,c, William Bentleyb,c, and Joseph Shiloacha

Abstract Background

Expression Verification Defining the Culture System

Conclusions

A vaccinia virus-mammalian cell expression system was developed as an alternative method for recombinant protein production utilizing EGFP as a reporter protein (1). In previous work, EGFP production was evaluated in T-flask culture and in both suspension and microcarrier based bioreactor systems, where general production parameters were defined. In this work, the production capability of the system, as defined with EGFP, was evaluated using two proteins, the HIV gp120 envelope glycoprotein and hGC-1 (2), an olfactomedin-related protein. These proteins contain complex post-translational modifications, required for gp120 activity and possibly for hGC-1, although there is little information on this recently discovered protein. Two recombinant vaccinia virus strains were engineered with the genes for gp120 or hGC-1 and expression of these proteins was achieved in either T-flask culture or both T-flask and bioreactor culture by infection of the cell culture with recombinant virus. The production process, purification protocol and glycosylation pattern of gp120 is described. Bioreactor culture produced secreted gp120 up to 40 mg/L at 66 hours post infection (hpi).

a Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bethesda, MD b Center for Biosystems Research, UMBI, College Park, MD c Department of Chemical Engineering, UMCP, College Park, MD

Production Parameters

gp120-6xHis Production gp120-6xHis Purification

Deglycosylation Analysis

Purification Methods

Deglycosylation Results

References

Based on reporter protein (EGFP) process development

• Cell Growth• 5 g/L Cytodex 3 microcarriers• 1.5x105 HeLa cell/mL initial seeding• DMEM+10% fetal bovine serum• 37°C, 30% dissolved oxygen, pH 7.0• Growth for 5 days to 1-2x106cell/mL

• Infection• 1 hour duration • MOI 5.0 (pfu/cell)• One third working volume• No serum in infection media (DMEM)• 1 mM IPTG added at infection

• Production• Return to 1.5 L volume and 10% serum• Reduce temperature to 34°C• Increase dissolved oxygen to 50%• 66 hours post infection

Clarify by centrifugation

48 hpi Reactor Supernatant

Load column with 50% slurry Ni-NTA resin (Qiagen) 1 mL resin per 100 mL supernatant

Wash column with base buffer (50 mM NaH2PO4300 mM NaClpH 8.0)

• Bind protein• Recycle supernatant through column for 4 hours

• Wash column • 1X with base buffer• 2X with base buffer + 20 mM imidazole

• Elute protein • Four fractions with base buffer + 200 mM imidazole

Treat to deactivate virus0.5% NP-40

500 mM NaH2PO4300 mM NaCl

Incubate overnight 4°C

• Vaccinia Virus• Orthopoxvirus family Poxviridae• Transcription occurs in cytoplasm of infected cell• Wide host range, includes most mammalian species and humans

• HeLa cells• Attachment dependent strain (ATCC CCL-2)• Microcarrier growth for larger cultures

• Enhanced Green Fluorescent Protein (EGFP)• Used as reporter protein to develop system parameters

• hGC-1• Olfactomedin-related protein (~64 kDa)• Potential for treatment of prostate and other cancers • Six N-linked glycosylation sites

• gp120• HIV envelope coat protein (~120 kDa)• Almost half the molecular weight attributed to N-linked glycosylations which are required for activity of the protein (3)

100 m

g/L gp

120

10 m

g/L gp

120

5 mg/L

gp12

0

1 mg/L

gp12

0

Prior t

o pur

ifica

tion

Flow th

roug

h

Was

h 1

Was

h 2

Was

h 3

Elution

1

Elution

2

Elution

3

Elution

4

100 m

g/L gp

120

10 m

g/L gp

120

5 mg/L

gp12

0

1 mg/L

gp12

0

Prior t

o pur

ifica

tion

Flow th

roug

h

Was

h 1

Was

h 2

Was

h 3

Elution

1

Elution

2

Elution

3

Elution

4

Coomassie Stained Gel Western Blot

250148

60

42

302217

64

kDa

Virus Construction

vT7lacOI* virus

HeLa Cells

Transfect

Infect

• Plaque isolation• Amplification• Viral purification• Viral titer

DonorPlasmid

Gene

pVOTE.2*

Plasmid

Gene

RecombinantPlasmid

Recombinant virus

* vT7lacOI virus and pVOTE.2 plasmid were kindly supplied by B. Moss and P. Earl, NIAID, NIH (4) and gp120 plasmid pTM-DHgp120H was kindly provided by M. Cho, School of Medicine, Case Western Reserve University (5).

Vacuum

LevelControl

FeedPump

WaterJacket

InletOutlet

DOpH

Agit.

Temp.

FiltratePump

AirInlet

MeshScreenModule

Diaphragm

ATFController

Gas inlet into reactor headspace

• HeLa cells (attachment dependent)• Microcarrier perfusion culture• 1.5 L working volume reactor

ATF™ System***

for microcarrier perfusion culture

*** ATF™ System was kindly supplied by Refine Technology, Co., East Hanover, NJ.

1. Bleckwenn, N.A., W.E. Bentley, and J. Shiloach, Biotechnology Progress, 2003. 19(1): p. 130-136.2. Zhang, J.C., et al., Gene, 2002. 283(1-2): p. 83-93.3. Hu, Y.C., et al., Biotechnology Progress, 2000. 16(5): p. 744-750.4. Ward, G.A., et al., Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(15): p. 6773-6777.5. Lee, M.K., M.A. Martin, and M.W. Cho, Aids Research and Human Retroviruses, 2000. 16(8): p. 765-775.

• Three different proteins were produced from three constructed viruses (EGFP, hGC-1, and gp120)• Growth, infection and production parameters were defined using reporter protein EGFP• gp120 was produced in a 1.5L bioreactor culture over 8 days with production for 66 hours

• Per liter of working volume• 4.7 mg secreted and purified gp120 was recovered at 48 hpi• 4 liters of media used• 200 mL serum used• 370 L viral stock used (1.9x1010 pfu/mL)

• Deglycosylation analysis of gp120 showed significant amount of N-linked glycosylations of approximately half the mass of the protein• Similar analysis will be performed on bioreactor culture with hGC-1 containing virus

EGFP ReporterProtein (intracellular)

hGC-1 Protein(intracellular)

gp120 Protein(extracellular)

Anti-FLAGantibody

250148

60

42

302217

64

kDaNeg

ative

contr

ol

+ hGC-1

-FLAG ge

ne

+ EGFP-6xHis gene Induced

+ EGFP-6xHis gene Not Induced

No virus

Flu

ores

cent

Mic

rosc

opy

Imag

es

** Anti-gp160 Antibody, HIV-1 gp160 Antiserum (HT3) from DAIDS, NIAID, NIH produced under contract by Repligen was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

Wes

tern

Blo

ts

Anti-gp160**

antibody

Negati

ve co

ntrol

+ gp12

0-6x

His ge

ne

250148

60

42

302217

64

kDa

Cleavage of N-linked SugarsPNGase F – Cleaves most N-linked oligosaccharides

(unless (1-3) core fucosylated)Cleavage of Common O-linked Sugars

O-Glycosidase – Cleaves O-linked unsubstituted Gal-(1-3)- GalNAc--

-2(3,6,8,9) Neuraminidase – Cleaves non-reducing terminal branched and unbranched sialic acids

Cleavage of less common hexasaccharide structures(1-4)Galactosidase – Cleaves (1-4)-linked, non-reducing terminal galactose

-N-Acetylglucosaminidase – Cleaves -linked N-acetylglucosamine

gp120 purified from reactor at 48 hpi in 10 mM Tris pH 7.0

Aliquot five fractions and treat sequentially with enzymes

1 2 3 4 5Treatment

Quantification achieved by scanning blot and comparing to gp120 standard**** with Scion Image software, Scion Corp.

18 66524844403024Time (hpi)

Western blot bands

****gp120 standard, HIV-1SF162 gp120 from Chiron Corporation and the DAIDS was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

Time (hpi)

0 10 20 30 40 50 60 70

Sec

rete

d g

p12

0-6x

His

(m

g/L

)

0

10

20

30

40

50

1.4x106 cell/mL infected at 0 hpi

gp12

0 stan

dard

Treatm

ent 1

Treatm

ent 2

Treatm

ent 3

Treatm

ent 4

Treatment 1 – No enzymesTreatment 2 – PNGase F Treatment 3 – PNGase F O-GlycosidaseTreatment 4 - PNGase F O-Glycosidase -2(3,6,8,9) NeuraminidaseTreatment 5 - PNGase F O-Glycosidase -2(3,6,8,9) Neuraminidase (1-4)Galactosidase-N-Acetylglucosaminidase

Treatm

ent 5

250

148

60

42

30221764

kDa

Std