vii
TABLE OF CONTENTS
CHAPTER
1
2
TITLE
PAGE
INTRODUCTION
1
1.1
Introduction
1
1.2
Objectives
3
1.3
Scopes of Research
3
LITERATURE REVIEW
5
2.1
Recombinant Protein Manufacturing Technologies
6
2.2
Glycosylation
8
2.2.1
N-Linked Glycosylation
9
2.2.2
O-Linked Glycosylation
9
2.3
Glycoprotein
10
2.4
Insect Cell- Baculovirus Expression System
11
2.4.1
Insect Cell Lines
11
2.4.2
Baculoviruses
12
2.4.2.1 Baculoviruses Replication
14
2.4.2.1.1 In Vivo Replication
14
2.4.2.1.2 In Vitro Replication
15
2.5
Advantages of BEVS Technology
20
2.6
Glycosylation in Insect Cells
22
viii
2.7
Glycosyltransferases and Glycosidases Involved
24
in N-glycan Processing in Insect Cells
2.7.1
α-Glucosidase I, II and α-Mannosidase I
24
2.7.2
N-Acetylglucosaminyltransferase I
25
(GlcNAcT-I) and α-mannosidase II
2.7.3
N-Acetylglucosaminyltransferase II
25
(GlcNAcT-II)
2.7.4
β-1,4-Galactosyltransferase (β-1,4GalT)
26
2.7.5
Core α-1,3- and α-1,6-Fucosyltransferases
26
(FucT)
2.8
2.7.6
β-N-Acetylglucosaminidase
27
2.7.7
Sialyltransferase (SiaT)
27
Sugar Nucleotides Involved in N-glycan
28
Processing in Insect Cells
2.8.1
Endogenous Sugar Nucleotide Levels
28
in Lepidopteran Insect Cells
2.8.2
Enzymes Involved in Sialic Acid and
28
CMP-Sialic Acid Synthesis
2.9
Engineering of N-glycan Processing Pathway
30
2.9.1
32
Improvement of N-Acetylglucosaminylation
of the Manα(1,3)- Branch
2.9.2
Improvement of Galactosylation
32
2.9.3
Production of Biantennary Complex-Type
33
N-glycans
2.10
2.9.4
Formation of Sialylated N-glycans
33
2.9.5
Synthesis of CMP-NeuNAc
33
Galactosylation in N-Glycan Processing in
34
Insect Cells
2.10.1 Sugar acceptor
35
2.10.2 Substrate Donor
35
2.10.3 Enzyme
38
ix
3
MATERIALS AND METHODS
41
3.1
Materials
41
3.2
Equipments
41
3.3
Chemicals
42
3.4
Spodoptera frugiperda (Sf-9) Insect Cells
43
3.4.1
Cells Thawing
43
3.4.2
Cells Maintaining
43
3.4.3
Cells Freezing
44
3.5
Wild Type and Recombinant Baculovirus
44
3.5.1
Virus Propagation
44
3.5.2
Virus Titrationr (End-Point Dilution)
45
3.5.3
Generating Pure Recombinant
46
Virus Stocks (End Point Dilution)
3.6
Recombinant Human Transferrin Detection
46
3.6.1
46
A
3.7
3.8
Sodium Dodecyl Sulfate – Polyacrylamide
Gel Electrophoresis
3.6.1.1 Silver Staining
47
3.6.2
Western Blot
48
3.6.3
Enzyme Linked Immunosorbent Assay
49
Recombinant β1,4-Galactosyltransferase Detection
49
3.7.1
Thin Layer Chromatography
49
3.7.2
Lectin Binding Assay
50
Native Uridine-5′-diphosphogalactose
51
(UDP-Gal) Level
3.8.1
UDP-Gal Extraction
51
3.8.2
Reverse Phase High Performance Liquid
52
Chromatography (RP-HPLC) Analysis
3.9
Coexpression of Recombinant Human Transferrin and
β1,4-Galactosyltransferase
52
x
4
RESULTS AND DISCUSSION
54
4.1
Sf-9 Cells Growth Optimization
55
4.2
Establishment of Baculovirus Expression
63
Vectors System (BEVS)
4.2.1
Mock Infection Optimization
63
4.2.2
Recombinant Human Transferrin
71
Expression
4.2.2.1 Time Course Expression of Recombinant
71
Human Transferrin
4.2.3
Recombinant β1,4-Galactosyltransferase
75
Expression
4.2.3.1 Time Course Expression of β1,4-
75
Galactosyltransferase
4.2.3.2 The Development of β1,4-
79
Galactosyltransferase Assay
4.2.4
Native Uridine-diphosphogalactose (UDP-Gal)
82
Monitoring at Normal and Upon Baculovirus
Infection
4.2.5
5
Baculovirus Coinfection Study
94
CONCLUSIONS
102
5.1
Conclusion
102
5.2
Further studies
104
REFERENCES
106
Appendices A-H
122
xi
LIST OF TABLES
TABLE NO.
4.1
TITLE
Growth Kinetics of Sf-9 Cells at Different
Parameters
PAGE
68
xii
LIST OF FIGURES
FIGURE NO.
2.1
TITLE
(a) N-linked protein glycosylation; (b) O-linked protein
PAGE
8
glycosylation
2.2
(a) High Mannose, (b) Complex and (c) Hybrid structures
10
of carbohydrates on the 3 major classes of glycoprotein
2.3
A few insect species used for glycoprotein production
11
2.4
Autographa californica multiple nuclear polyhedrosis virus
13
(AcMNPV)
2.5
A) Baculovirus particles, or polyhedra; B) Cross-section of
13
a polyhedron; C) Diagram of polyhedron cross-section.
(Jean Adams and V. D'Amico.)
2.6
In vivo baculovirus infection and replication
14
2.7
In vitro baculovirus infection and replication
17
2.8
Structural compositions of the two baculovirus phenotypes,
18
budded virus (BV), and the occlusion derived virus (ODV)
(Blissard, 1996)
2.9
a) A typical infected Sf-9 cells ( Steven Howard);
19
(b) Electron micrograph of AcMNPV infected Sf-9 Cell
(Greg V.Williams); (c) A portion of the nucleus containing
enveloped virions in the process of being occluded into a
developing polyhedron (Queen's University)
2.10
Protein N-glycosylation pathways in insect and mammalian
cells
23
xiii
2.11
CMP-Neuraminic acid synthesis pathway
29
2.12
General strategy for humanization of glycoprotein produced
31
by lepidopteran cell-baculovirus expression system
2.13
Structure of a nucleotide sugar that can serve as a sugar
37
donor in a glycosyltransferase reaction
2.14
Transporters for sugar nucleotides, PAPS, and ATP are
37
located in the Golgi membranes of mammals, yeast,
protozoa, and plants
3.1
Virus Titer Procedures – End Point Dilution
45
4.1
Sf-9 insect cells growth in monolayer culture at 3 different
56
serum concentrations. (a) TC-100 and (b) SF-900 II SFM
4.2
Sf-9 insect cells growth in monolayer culture for 2 types
58
of media. (a) without serum;(b) with 5% serum and
(c) with 10% serum
4.3
Sf-9 insect cells growth in monolayer culture for 3 different
59
initial cell density, i.e. 0.2, 1.2 and 2.33 x 105 cells/ml
4.4
Sf-9 insect cells growth in monolayer culture at 3 different
61
subculturing conditions, i.e. early exponential, late exponential
and stationary phase
4.5
Sf-9 insect cells growth in monolayer culture at 3 different
63
spent medium carry over percentage, i.e 100%, 50% and 0%
4.6
The effect of initial cell density on Sf-9 insect cells infected
64
with wild type AcMNPV viruses at MOI 10
4.7
The effect of spent medium carry over on Sf-9 insect cells
65
infected with wild type AcMNPV viruses at MOI 10
4.8
The effect of MOI on Sf-9 insect cells infected in the
67
stationary phase with wild type AcMNPV Viruses
4.9
(a) 9% SDS-PAGE analysis with silver stained; (b) Western
72
blot analysis indicated the rhTf protein synthesized in Sf-9
cells supernatant at hour 120
4.10
Time Course of rhTf protein production in supernatants
73
were resolved on 9% SDS-PAGE and stained with silver
4.11
Time course of rhTf protein production in (a) Lysates;
(b) Supernatants were detected using ELISA.
74
xiv
4.12
Detection of β1,4-GalT by using chromatogram of
76
thin layer chromatography
4.13
Time course of chromatogram of thin layer chromatography.
77
4.14
SDS-PAGE (9%) Time Course of β1,4-GalT Production
78
4.15
Standard curve for the determination of β1,4-GalT
80
activity from the Lectin Binding Assay values
4.16
Time course of β1,4-GalT enzyme production in
81
supernatants were detected using lectin binding assay.
4.17
RP-HPLC chromatogram for UDP-Gal standard at different
84
concentration
4.18
Standard curve for UDP-Gal
85
4.19
RP-HPLC chromatogram for native UDP-Gal sample with
87
spiking and without spiking
4.20
RP-HPLC Chromatogram for the time course of native
88
UDP-Gal level upon infection time at (a) 0h (Normal);
(b) 24h; (c) 48h; (d) 72h; (e) 96h and (f) 120h (Set Data 1)
4.21
RP-HPLC chromatogram for time course of native
89
UDP-Gal level upon infection in 3D diagram (Set Data 1)
4.22
RP-HPLC Chromatogram for the time course of native
90
UDP-Gal level upon infection time at (a) 0h (Normal);
(b) 24h; (c) 48h; (d)72h; (e) 96h and (f) 120h (Set Data 2)
4.23
RP-HPLC chromatogram for time course of native
91
UDP-Gal level upon infection in 3D diagram (Set Data 2)
4.24
Native UDP-Gal concentration in µM at normal and upon time
92
of infection
4.25
Verification of UDP-Gal fractions from RP-HPLC
93
analysis by using chromatogram of TLC
4.26
Galβ1→4GlcNAc linkage binding values at 450nm
for the time course upon coinfection between recombinant
baculovirus hTf and β1,4-GalT
96
xv
4.27
Effect of the mammalian galactosyltransferase on the
97
rate of in vitro galactosylation process
4.28
Galβ1→4GlcNAc linkage binding values at 450nm for the
100
different level of galactosylation process
4.29
Relationships among the three main elements in in vivo
galactosylation process
101
xvi
LIST OF SYMBOLS/ ABBREVIATIONS
2-ADN
-
2-acetamide-1,2-dideoxynojirimycin
AcMNPV
-
Autographa californica multicapsid nucleopolyhedrovirus
Asp
-
Asparagine
Ba(OH)2
-
barium hydroxide
BEVS
-
baculovirus expression vectors system
bIFN- γ
-
bovine interferon-γ
Bm
-
Bombyx mori
BSA
-
bovine serum albumin
BVs
-
budded viruses
CaCl2
-
calcium chloride
CHO
-
chinese hamster ovary
CMP
-
cytidine-5’-monophosphate
CMP-NeuNAc-
cytidine-5’-monophospho N-acetylneuraminic acid
CMP-SAS
-
CMP-NeuNAc synthase
DMSO
-
dimethyl sulphoxide
DNA
-
deoxyribonucleic acid
E.Coli
-
Escherichia coli
Ea
-
Estigmene acrea
xvii
EDTA
-
ethylenediamine tetraacetic acid disodium salt dehydrate
ELISA
-
Enzyme Linked Immunosorbent Assay
ER
-
endoplasmic reticulum
FBS
-
fetal bovine serum
Fuc
-
fucose
FucT
-
Fucosyltransferases
Gal
-
galactose
GalNAc
-
N-Acetylgalactosamine
GDP-Fuc
-
guanosine 5’-diphoshate-β-L-fucose
GDP-Man
-
guanosine 5’-diphoshate-D-mannose
Glc
-
glucose
GlcNAc
-
N-Acetylglucosamine
GlcNAcT II
-
N-Acetylglucosaminyltransferase II
GlcNAcT-I
-
N-Acetylglucosaminyltransferase I
H2O2
-
peroxidase
H3PO4
-
phosphoric acid
HCl
-
hydrochloric acid
HRP
-
horseradish peroxidase
hTf
-
human serum transferrin
IgG
-
immunoglobulin G
kbp
-
kilobasepairs
kDa
-
kilodalton
LacNAc
-
N-Acetyllactosamine
M
-
molar
Man
-
mannose
ManNAc
-
N-Acetylmannosamine
ManNAc
-
N-acetylmannosamine
ManNAc-6-P -
N-acetylmannosamine-6-phosphate
MB
-
Mamestra brassicae
Mg
-
magnesium
min
-
minute
mm
-
mililiter
MnCl2
-
manganese chloride
MOI
-
Multiplicities of Infection
xviii
MOPS
-
4-Morpholinepropanesulfonic acid
MWCO
-
molecular weight cut off
NaCl
-
sodium chloride
NAG
-
N-acetylglucosamine
NAL
-
N-Acetyllactosamine
NeuNAc
-
N-acetylneuraminic acid
NeuNAc-9-P -
N-Acetylneuraminic acid-9-phosphate
nm
-
nanometer
NOV
-
non-occluded virus particles
NPV
-
nucleocapsid nuclear polyhedrovirus
OBV
-
occlusion body-derived virus particles
PBS
-
Phosphate Buffer Saline
PBST
-
PBS containing 0.05% Tween 20
PI
-
Post infection
RCA I
-
Ricinus communis agglutinin 1
RP-HPLC
-
Reverse Phase High Performance Liquid Chromatography
rpm
-
rotation per minutes
SAS
-
N-Acetylneuraminate-9-phosphate synthase
SDS
-
sodium dodecyl sulfate
SDS-PAGE
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Sf
-
Spodoptera frugiperda
SiaT
-
sialyltransferase
TBAS
-
tetrabutylammonium hydrogen sulfate
TBS
-
Tris-buffered Saline
TCID50
-
Tissue Culture Infectious Dose 50
TEMED
-
N,N,N',N'-tetramethylethylenediamine
TLC
-
Thin Layer Chromatography
TMB
-
3,3’,5,5’-tetramethylbenzidene
Tn
-
Trichoplusia ni
TOI
-
Time of infection
UDP-Gal
-
uridine-diphosphogalactose
UDP-GlcNAc -
uridine-5’-diphopho-N-acetylglucosamine
UDP-hexose -
uridine-5’-diphopho-D-hexose
UF
ultrafiltration
-
xix
UTP
-
uridine 5’-triphosphate sodium
UV
-
ultraviolet
ZnSO4.7H2O -
zinc sulfate 7-hydrate
α2,6-ST
-
α2,6-sialytransferase
β1,4-GalT
-
β1,4-galactosyltransferase
µl
-
microliter
µm
-
micrometer
0
-
degree Celcius
C
xx
LIST OF APPENDICES
APPENDIX
TITLE
PAGE
A-1
Monosaccharide Mass and Structure
122
A-2
Common N-Linked Glycan Simplified
123
Structures and Masses
A-3
Cell Culture Glossary
124
B-1
Stock Solution for SDS-PAGE
126
B-2
Working Solution for SDS-PAGE
127
B-3
Separating and Stacking Gel Preparation
128
C
Working Solution for ELISA
129
D
Working Solution for Western Blot
130
E
Virus Calculation
131
F
Reaction Mixture for Lactose Synthetase
132
Assay
G
Reaction Mixture for Lectin Binding Assay
135
H
Publications
136