Production and Glycosylation Analysis of Model Proteins
from a Vaccinia Virus-Mammalian Cell Expression System
Nicole A.
a
a,b,c
Bleckwenn ,
William
b
Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bethesda, MD
Abstract
hGC-1 Protein
(intracellular)
Western Blots
Fluorescent Microscopy Images
+ EGFP-6xHis gene
Induced
60
42
42
30
22
17
30
22
17
+ EGFP-6xHis gene
Not Induced
Vacuum
Mesh
Screen
Module
Anti-FLAG
antibody
Water
Jacket
*** ATF™
Secreted gp120-6xHis (mg/L)
40
30
10
0
50
60
70
Wash column with
base buffer
(50 mM NaH2PO4
300 mM NaCl
pH 8.0)
Time (hpi)
Western blot bands
Quantification achieved by scanning blot and comparing to gp120
standard**** with Scion Image software, Scion Corp.
****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.
Deglycosylation Analysis
1
2 3 4 5
Treatment
ATF
Controller
Air
Inlet
pVOTE.2*
Plasmid
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).
Production Parameters
for microcarrier
perfusion culture
Cleavage of N-linked Sugars
PNGase F – Cleaves most N-linked oligosaccharides
(unless a(1-3) core fucosylated)
Cleavage of Common O-linked Sugars
O-Glycosidase – Cleaves O-linked unsubstituted Gal-b(1-3)GalNAc-aa-2(3,6,8,9) Neuraminidase – Cleaves non-reducing terminal
branched and unbranched sialic acids
Cleavage of less common hexasaccharide structures
b(1-4)Galactosidase – Cleaves b(1-4)-linked, non-reducing
terminal galactose
b-N-Acetylglucosaminidase – Cleaves b-linked Nacetylglucosamine
Treat to deactivate virus
0.5% NP-40
500 mM NaH2PO4
300 mM NaCl
Incubate overnight 4°C
Deglycosylation Results
kDa
250
148
Std
60
42
30
22
17
6
4
• 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
gp120-6xHis Purification
48 hpi Reactor
Supernatant
• 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
Time (hpi) 18 24 30 40 44 48 52 66
Aliquot five
fractions and treat
sequentially with
enzymes
Recombinant
virus
* vT7lacOI
Purification Methods
20
gp120 purified from
reactor at 48 hpi in
10 mM Tris pH 7.0
Donor
Plasmid
ATF™ System***
Clarify by
centrifugation
40
Gene
System was kindly supplied by Refine Technology, Co., East Hanover, NJ.
Load column with 50%
slurry Ni-NTA resin
(Qiagen) 1 mL resin per
100 mL supernatant
cell/mL infected at 0 hpi
30
• Plaque isolation
• Amplification
• Viral purification
• Viral titer
Recombinant
Plasmid
antibody
50
20
Gene
Inlet
Outlet
gp120-6xHis Production
10
Transfect
• HeLa cells (attachment dependent)
• Microcarrier perfusion culture
• 1.5 L working volume reactor
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.
0
HeLa Cells
Gas inlet
into reactor
headspace
Feed
Pump
** Anti-gp160 Antibody,
1.4x106
Filtrate
Pump
DO
pH Temp.
Agit.
Level
Control
Anti-gp160**
Infect
Based on reporter protein (EGFP) process development
6
4
6
4
Department of Chemical Engineering, UMCP, College Park, MD
vT7lacOI*
virus
gp120 Protein
(extracellular)
kDa
250
148
60
a
Shiloach
Virus Construction
Defining the Culture System
Diaphragm
No virus
c
Center for Biosystems Research, UMBI, College Park, MD
• 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)
Expression Verification
kDa
250
148
and Joseph
Background
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).
EGFP Reporter
Protein (intracellular)
b,c
Bentley ,
Treatment 1 – No enzymes
Treatment 2 – PNGase F
Treatment 3 – PNGase F
O-Glycosidase
Treatment 4 - PNGase F
O-Glycosidase
a-2(3,6,8,9) Neuraminidase
Treatment 5 - PNGase F
O-Glycosidase
a-2(3,6,8,9) Neuraminidase
b(1-4)Galactosidase
b-N-Acetylglucosaminidase
Coomassie Stained Gel
Western Blot
kDa
250
148
60
42
30
22
17
6
4
Conclusions
• 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 mL viral stock used (1.9x1010 pfu/mL)
• Deglycosylation analysis of gp120 showed significant amount of Nlinked glycosylations of approximately half the mass of the protein
• Similar analysis will be performed on bioreactor culture with hGC-1
containing virus
References
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.