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Generation of mutant tissue inhibitor of metalloproteinases-2 (TIMP-2) in the baculovirus expression system
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Generation of mutant tissue inhibitor of metalloproteinases-2 (TIMP-2) in the baculovirus expression system
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GENERATION OF MUTANT TISSUE INHIBITOR OF
METALLOPROTEINASES-2 (TIMP-2) IN THE
BACULOVTRUS EXPRESSION SYSTEM
By
Stephen Triumph-Shien Kwan
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment o f the Requirements for the Degree
Master of Science
(Biochemistry and Molecular Biology)
COPYRIGHT 1998 Stephen Triumph-Shien Kwan
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UMI Number: 1393175
UMI Microform 1393175
Copyright 1999, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized
copying under Title 17, United States Code.
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U N IV E R S IT Y O F S O U T H E R N C A L IFO R N IA
T H E GRADUATE SCH O O L
U N IV ER SITY PARK
LOS A N G E LE S. CA LIFO R N IA S 0 0 0 7
This thesis, •written by
S r e w E N H ?xuH ph - S h i& v I ( ^ a / /
under the direction of his, Thesis Committee,
and approved by a ll its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
«
cr~-
D a te -^&ar —
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O X Q Q / f ( i c / t e l l e j Q ^ c d w t , cmd G £ $ ea ; .
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ACKNOWLEDGEMENTS
I would like to take this opportunity to express my greatest gratitude toward my
mentor and advisor, Dr. Yves DeClerck, for all his guidance, encouragement and
enthusiasm for supporting my master thesis.
It was a long year, I am greatly indebted to all my lab colleagues for their advises
and suggestions, Laurence Blavier, Patrick Henrit, Yasuo Sangauri, Zhi-Duan Zhao and
especially Patrick Sato who helped me started the baculovirus expression system.
I am grateful to numerous numbers o f researchers and staffs at CHLA who had
helped me directly or indirectly leading to the completion o f this thesis.
Also special thanks to Jim Luo o f Multi-Image Group for his extraordinary
technical support and computer facility.
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CONTENTS
LIST OF THE FIGURES
LIST OF THE TABLES
ABSTRACT
INTRODUCTION
1. Introduction o f TIMPs.
2. The Structure o f TTMP-2.
3. Multifunctional Ability of TIMP-2.
4. The Rationale o f the Generation o f Mutant TIMP-2.
5. Introduction to Baculovirus Expression System.
6. The Advantages o f Baculovirus Expression System.
7. Insect Cell Lines.
MATERIALS AND METHODS
1. BlueBacHis 2B Expression Vector.
2. PCR Product Analysis.
3. AcMNPV (Bac-N-Blue)
4. Sf-9 Cell Line.
5. High-Five Cell Line.
6. Baculoviruse Agarose for plaque assay.
7. Primers used to detect the presence o f TIMP-2 cDNA within the Baculovirus
Expression Vector.
8. Western Blot Analysis.
RESULTS
1. Generation o f rTTMP-2 1-101, Cysl3Ala.
2. Sequencing o f rTIMP-2 1-101, Cysl3Ala.
3. Transfection into the Sf-9 cells.
4. Plaque Assay.
5. PCR Analysis o f Recombinant Baculovirus.
6. Gemation o f High-Titer Recombinant Viral Stocks.
7. Time Course Expression.
DISCUSSIONS
REFERENCES
l
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LIST OF THE FIGURES
Figure - 1: The Structure o f TIMP-2.
Figure - 2: The Rationale for the Generation o f Mutant TIMP-2.
Figure - 3: Insect Cells.
Figure - 4: PCR Generation o f TIMP-2 1-101, Cysl3Ala Mutant.
Figure - 5: Construction o f TIMP-2 1-101, Cysl3Ala Mutant.
Figure - 6: Sequence Confirmation.
Figure - 7: Liposomal Cotransfection o f Sf-9 Insect Cells.
Figure - 8: Stages o f Transfection.
Figure - 9: PCR Analysis o f Recombinant Virus.
Figure - 10: Tim e Course Analysis o f TIMP-2 1-101, Cysl3A la Expression in Sf-9 Cells.
Figure - 11: Time Course Analysis o f TIMP-2 1-101, Cysl3Ala Expression in High-Five
Cells.
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LIST OF TABLES
Table #1:
Table #2:
Table #3:
Table #4:
Table #5:
Table #6:
Table #7:
Table #8:
Characteristics o f TTMP Family.
The Functional Domains o f TIMP-2.
Preliminary W ork on Cys-Ala Mutations.
Comparison Between Baculovirus and Bacterial System.
Observations o f the Stages o f Transfection.
P -l Recombinant Viral Stock Titer.
P-3 Recombinant Viral Stock Titer.
Time Course Expression with Different MOI by Infecting High-Five cells.
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ABSTRACT
Tissue inhibitors o f metalloproteinases (TIMPs) are a family o f natural inhibitors
o f matrix metalloproteinases (MMPs), a class o f proteases that degrade a wide range o f
extracellular matrix components. TIMP-2 is a MMP inhibitor that has been particulary
studied and was cloned and identified in our laboratory.
In addition to its anti-MMP activity, TIMP-2 has also been shown to bind to the
cell surface and modulate cell growth. To obtain a better insight into this dual function of
TIMP-2, a baculovirus expression system was used to generate TIMP-2 mutants.
A TEMP-2 1-101, Cysl3Ala cDNA, in which 93 amino acids in the C-terminal
region were deleted and cystine at position 13 was substituted by alanine, was generated
by PCR and inserted into the pBlueBacHis 2B expression vector. The plasmid was
propagated in DH5a cells and cotransfected with the linearized type AcMNPV DNA by
liposomal transfection. The recombinant-AcMNPV-TIMP-2 1-101, Cysl3A la viruses
were isolated by plaque assay and used to infect Sf-9 cells to produce high-titer
recombinant virus. The success o f generating recombinant TIMP-2 1-101, Cysl3Ala
virus was confirmed by the overlay agarose plaque assay and a titer o f 2.0 X 108 pfu/ml
was obtained. Time course analysis of the recombinant mutant rTIMP-2 expression was
performed by infecting Sf-9 and High-Five insect cells and the recombinant protein was
detected by Western blot. Different sets o f multiplicity o f infection were applied in order
to study the kinetics, and to determine the optimal expression time for the recombinant
protein.
4
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The TIMP-2 1-101, Cysl3A la protein generated by baculovirus expression
system will be purified and examined for its antimetalloproteinase and growth promoting
activities. These studies will provide a deeper knowledge on the dual function o f TIMP-2
and helpful information to locate the domain o f TIMP-2 that interact with the active site
o f MMPs and cell surface associated putative receptor.
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INTRODUCTION
The m ost widely appreciated biological function o f the tissue inhibitors o f
metalloproteinases (TIMPs) in cancer is their role associated with the inhibition o f cell
invasion in vitro(l,2), tumorigenesis(3,4) and metastasis, in vivo(5,6,7). Since the net
matrix metalloproteinase (MMP) activity is the result o f the balance between the
activated enzyme levels and TIMP levels, an increase in the amount o f TIMPs relative to
MMPs could function to block tumor cell invasion and metastasis. The underlying
molecular mechanism for the tumor suppressing activities o f TIMPs, nevertheless, is
thought to depend on their ability to inhibit active MMP directly and to regulate the
autoactivation process o f proMMP (8,9,10). In addition, TIMP-2 has been demonstrated
to have an anti-angiogenic activity, and such inhibition of angiogenesis is mediated by
inhibition o f both endothelial cell proliferation and migration (11).
The TIMP gene family has 4 members with 30-40% amino acid sequence
homology. A consensus sequence CXCXPXHPQXAFCNXDXVERAK (X = any amino
acid) at the N-terminus was identified based on the genetic and biochemical data (12). In
addition, TIMPs contains 12 conserved cysteines forming critical intramolecular disulfide
linkages into a total of six disulfide knots. All members o f TIMP family have a similar
inhibitory ability toward all MMPs, with one exception, MT1-MMP which is inhibited by
TIMP-2 and 3, but not TIMP-1 (13). The TIMP family protein inhibits the proteolytic
activity o f MMPs by forming a tight 1:1 stoichiometric nonco valent complex (Ki at
nanmolar range.)(14) Although the members o f TIMPs have very similar inhibitory
6
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activities against most members o f MMP family, TIMPs differ in various aspects such as
solubility, glycosylation, regulation, specific tissue expression, location on human
chromosome, and isoelectric points (Table #1) (15,16,17,18).
Table #1 Characteristics of TIMP Family
Characteristics TTMP-1 TIMP-2 TIMP-3 TTMP-4
Mol. Weight 28 kDa 21 kDa 24 kDa 23 kDa
Glycosylation Yes No Yes No
Leader Sequence 24 aa 26 aa 24 aa 24 aa
Preferential
Binding to
proMMP
ProMMP-9 ProMMP-2 ProMMP-2
ProMMP-9
ProMMP-2
Solubility Soluble Soluble Binds to ECM Soluble
Regulation Upregulated
by TP A &
TGF
Constitutive
Expressed
Upregulated by
TPA & TGF
Tissue
Expression
Bone, ovary,
heart
Lung, muscle,
brain, skin,
reproductive
organs.
Kidney, heart,
lung, ovary
Heart, muscle,
brain
Chromosome
Location
(Human)
X 17 22
?
Isoelectic Point 8.00 6.45 9.04 7.34
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A B
ICys 1-Cys 72
\ 2 V ' X
if 3 \
y * ■
••••• 4 •*
• • • • •
• •
• • • • • • • • • • •
N-terminus
Cys 3-Cys 101
alpha helix
/
C-terminus
Cys iS-Cys 126
N-terminus
Figure-1. The Structure of TIMP-2
A. Primary Structure of TIMP-2
TIMP-2 consisted o f two globular domain, N-and C-terminus. Each domain encompassed three interlinked disulfide bonds
forming a total of 6 loops. This primary structure of TIMP-2 was based on the assignment of the disulfide bonds according to
Williamson et al.(22)
B. Tertiary Structure of N-Terminal Generated by MolScript Representation
This is a reproduction of the MolScript representation of tertiary structure of N-terminal domain of TIMP-2 (22). The 6 p-sheets
were labeled from A to F and a-helices were labeled as 1 and 2, which were determined by 'H NMR 3D NOESY-TOCSY data (23).
The ball-and-stick representation demonstrated the links of the Cysl-Cys72, Cys3-Cysl01 and Cys 13-Cys 126 disulfide bonds formed
a 3 disulfide knots shown on Figure A above.
00
The Structure o f TEMP-2
TIMP-2 consists of two globular domains, N - and C- terminus. Each domain
comprises three interlinked disulfide bonds form ing a total o f six loops (Figure-1). The
N-terminal region (residues 1 to 22) o f the TIMP family is highly conserved and thus
may contribute to the inhibitory activities, and the C-terminal region is more divergent
and may influence the selectivity toward the target enzymes. It is known that different
members o f TIMP family have its preferential binding to the proMMPs. TTMP-l forms a
preferential complexes with proMMP-9 (19), and TIMP-2 and TIMP-4 preferentially
complex with proMMP-2 (20,21).
* 1 1 MNR 3D NOESY-TOCSY study reveals that the N-terminal domain o f TIMP-
2 has an OB (oligosaccharide/oligonucletide binding) folding motif, with 6 ( 3 sheet and
five o f them placed in an antiparallel structure fashion forming a closed (3-barrel (22).
Figure-IB is a reproduction o f the MolScript representation o f the tertiary structure o f N-
terminal domain o f TIMP-2. The 6 (3-sheets were labeled from A to F and a-helices were
labeled as 1 and 2. The ball-and-stick representation demonstrated the links o f the Cysl-
Cys72, Cys3-Cysl01 and Cys 13-Cys 126 disulfide bonds formed a 3 disulfide knots.
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Multifunctional Ability o f TIMP-2
TIMP-2 is a multifunctional protein, which acted as an inhibitor o f matrix
metalloproteinases with its antimetalloproteinase activity and also can acted as a growth
stimulus on normal and abnormal cells with its ligand-binding ability (24).
Many mutagenesis studies (23,24,25) have indicated that the N-terminal domain
o f TIMP-2 is responsible for the antimetalloproteinase activity, but peptide fragment
analysis on TIMP-1 indicated that there is no particular amino acid solely responsible for
such activity (26). The antimetalloproteinase acitivity o f the N-terminal has also been
shown with its inhibition with several MMPs, including intestitial collagenase(27),
stromelysin-1, gelatinase A, and MMP-7 (28,29)
Elucidation o f the function o f the C-terminal is currently under investigation. It is
known that C-terminal domain o f TIMP-2 plays as a major role in the interaction o f
TIMP-progelatinase complex (29, 30). It is hypothesized that the C-terminal domain o f
TIMP-2 bind to the C-terminal domain o f progelatinase A to allow the N-terminal
domain o f TIMP-2 to interact with active MMPs (31).
Table #2 The Functional Domains of TIMP-2
Functions N-terminal Domain C-terminal Domain
Inhibition o f active MMP
V
—
Binding to proMMP-2 to modulate the
autoactivation V
Binding to cell surface to promote growth
o f the erythroid precursor cells
?
V
(23-31)
10
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* 2
• • • • • •• ’•
U 3 \ \ 2 \ * 2 *
. • 5 1 / 3 \ * . . . * |
'* \ V 4 ) / \ **..
• * . ....... •. v
6 i f ’• / • t
a a w a • m A a
\ • * 6 / f • f
i \ ' - ' I ’ \ [
• * •« / *•__
A. Full Length TIMP-2 B . TIMP-2 1 -128,Cysl 3Ala Q, TIMP-2 1-101, Cysl 3Ala
Figure -2 The Rationale for the Generation of Mutant TIMP-2
A. Full Length TIMP-2.
B. TIMP-2 1-128, Cys 13Ala was generated by PCR from pCDNAHMI (HMI is identical
to TIMP-2.)
C. To further characterize and analyze the essential role of the first and second disulfide
bonds in maintaining the functional structure of TIMP-2, baculovirus expression
system was used to generate a recombinant TIMP-2 1-101, Cys 13 Ala (truncated at
position 102 and site specific mutated at Cysl3.) from the template TIMP-2 1-128,
Cys 13 Ala.
The R ationale fo r the G eneration o f M u tan t TIM P-2 (Fig-2)
The preliminary works from DeClerck lab had generated 3 rTTMP-2 mutants, in
which Cys were individually replaced by Ala in order to discover the importance o f each
disulfide bond in the N-terminal domain, by transfected into the CHO cells. The analysis
o f antimetalloproteinase activity o f these mutants was determined by reverse zymography
which test the retention o f antimetalloproteinase activity (Table #3.)
Table #3 Prelim inary W o rk on Cys-Ala M utations
Location o f Cys - Ala M utations Retention o f antimetalloproteinse activity
Cys 13 Yes
Cys 72 No
Cys 101 No
The result o f the Cys-Ala mutations experiment indicated that first and second
disulfide bonds were required in maintaining the antimetalloproteinase activity and the
substitution mutation at Cys 13, had no effect on the antimetalloprotinase activity of
TIMP-2 (Table #3.) To further characterize and analyze the essential role o f the first and
second disulfide bonds in maintaining the functional structure o f TIMP-2, baculovirus
expression system was used to generate a recombinant TTMP-2 1-101, Cys 13 Ala
(truncated at position 102 and site specific mutated at Cys 13.)
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Introduction to Baculovirus Expression System
The baculovirus expression system is a powerful and versatile eukaryotic
expression system. This system is applied to express heterologous genes from various
species including mammal, plant, fungi, and bacterial in insect cells.
Baculoviruses are diverse group o f double stranded DNA viruses, and highly
specific in infecting various insect species. Autographa califomica multiple nuclear
polyhedrosis virus (AcMNPV) is a most extensively studied baculovirus strain, which
was fully mapped and sequenced (32,33) The commercialized AcMNPV (Bac-N-Blue®)
has the polyhedrin gene deleted, to allow the desired heterologous cDNA to be inserted
into it. The polyhedrin gene has been shown to be nonessential for infection or
replication o f the virus in tissue culture setting (34). Biologically, the polyhedrin protein
is a major structural component o f the viral occlusions, providing a mean for horizontal
transmisssion o f the virus during harsh conditions (35).
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The Advantages o f Bacnlovirus Expression System (Table #2)
The major advantage o f baculovirus expression system is the production o f
functional active recombinant proteins. Since the major drawback in using bacterial
systems is the fact that the recombinant proteins expressed are usually insoluble,
aggregated (forming inclusion body), incorrectly folded and lack the post-translational
modification that is important for many proteins. A major advantage o f using baculovirus
expression system is the fact that the expressed recombinant proteins are properly folded,
and the formation o f disulfide bonds is maintained., and post-translationally modified
(phosphorylation, glycosylation, and acylation). (33,34,36)
Table #4 Comparison Between the Features o f Baculovirus and Bacterial System
Features Baculovirus System Bacterial System
Post-translation modification Phosphorylation,
Glycosylation, Acylation
Phosphorylation
Intron splicing Yes No
Protein size Not restricted Less than 100 kDa
Multiple gene expression Yes No
Nuclear transport Yes No
Functional protein Yes Sometimes
Direct cloning Yes Yes
(33,34,35,36,37)
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F1g.-3 INSECT CELL LINES USED IN BACULOVIRUS EXPRESSION SYSTEM
A. Log Phase Sf-9 Insect Cells
Sf-9 insect cells grown in TNM-FH medium. The adherent culture was maintained at
a cell density o f 1.0 X106 cells/ml and passaged by sloughing dislodge method at its
confluency.
B. Log Phase High-Five Insect Cells
High-Five insect cells grow well in Excell 400 (JRH Bioscience), serum-free
medium, ft has a very similar characteristic as Sf-9 cells, but it uses glucose as the
source of energy and serum in not required for growth. The adherent culture was
maintained at a cell density of 1.5 X 105 cells/ml and passaged by slougjhing dislodge
method at confluency.
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Insect Cell Lines (Figure-3)
The m ost popular insect cell lines used in baculovirus expression system are Sf-9
(Spodoptera frugiperda), Sf-21, and High-Five cells. All o f them have a doubling time
less than 24 hours, a spherical morphologic, and a loosely attachment to the surface.
The insect cell lines used in our experiments were Sf-9 and High-Five cells. The
Sf-9 cell line is derived from the pupal ovarian tissue o f the fall army worm (38). The Sf-
9 cells have a doubling time o f about 20 hours and grow in both monolayer on tissue
culture dish and suspension in spinner flask.
The High-Five insect cell line (BT1-TN-5B1-4) was originally developed by the
Boyce Thompson Institute, and originated from the egg cells o f the cabbage looper,
Trichoplusia ni. High-Five cells grow well in Excel 400 serum-free medium. The High-
Five cells are very similar to Sf-9 cell in term o f their characteristics and features. A
significant advantage o f this cell line is that they grown in serum-free medium, double in
less than 20 hours and can provide up to 10-fold higher levels o f secreted recombinant
protein than Sf-9 cells (39,40)
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MATERIALS AND METHODS
BlueBacHls 2B Expression Vector
The pBlueBacHis 2B constructed by Invitrogen has an ATG start codon at
position 95 and an Ampicillin resistance gene at position 1643. An Enterokinase
Recognition Site is located at position 176, and can be recognized by Anti-Xpress
Antibody (Invitrogen) allowing to identify the recombinant protein immunologically. The
recombination sequences at bases 456-1256 has an Open-Reading Frame 1629 (ORF
1629) allowing the recombination with AcMnPV DNA to generate a recombinant
baculovirus. The 5’ lacZ fragment at 3439-4551 which encodes B-galatosidase, an
enzyme that cleave X-gal (5-bromo-4-chloro-3-indoly-(5-D-galactose), serves as a
selection tool in plaque assay and is used to determine the titer o f the recombinant
viruses. The ColEl origin allows high-copy number replication and growth in DH5a
competent cell. The 6X Histidine region codes for six histidine residues, which allow the
expressed recombinant protein to bind to a nickel affinity column (35). The multiple
cloning site at position 190-245, is composed of various restriction sites allowing the
insertion o f rTIMP-2 1-101, Cysl3Ala cDNA.
PCR Product Analysis
The size o f the PCR products and pBlueBacHis 2B was determined by loading
samples into 1% agarose in TAE buffer (40 mM Tris-acetate, pH 7.8; 5 mM sodium
acetate, 1 mM EDTA and 1 pg/ml ethidium bromide) and running the electrophoresis at
100 V for one hour. The concentration o f the PCR product was determined by
spectrometry at 260 nm wavelength before loading into the agarose.
17
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AcMNPV (Bac-N-BIue®)
AcMNPV (Bac-N-Blue®) had polyhedrin gene deleted, to allow the desired
heterologous genes to be inserted into it. Circular Bac-N-Blue DNA® from Invitrogen,
was restriction digested by Bsu36 which e liminated part o f the polyhedrin gene and ORF
1629 to enhance the recombination process. During the liposomal cotransfection, with the
aid o f InsectionPlus® liposomes, linearized Bac-N-Blue DNA was allowed to recombined
with pBlueBacHis 2B-1IM P-2 1-101, Cysl3Ala with two possibilities to generated a
recombinant AcMNPV - TEMP-2 1-101, Cysl3Ala viruses. The polyhedrin promoter
( P p h ) was a strong promoter located at the upstream o f inserted TIMP-2 l-101,Cysl3A la
cDNA for driving an efficient expression. The P e t l promoter, adjacent to P p h was used to
drive the expression o f lacZ gene, in another direction, to produce (3-galactosidase.
Sf-9 Cell Line
Sf-9 insect cells were grown in TNM-FH medium, which consisted o f Grace's
Insect Media(Invitrogen), lactalbumin hydrolysate, L-glutamine, TC-yeastolate, 10%
fetal bovine serum (Gemini), and gentamycin (10 pg/ml). The adherent culture was
seeded at a cell density o f 1.0 X 106 cells/ml and passaged by sloughing dislodge method
at its confluency. This insect line was used in transfection experiment, plaque
purification, the generation o f high-titer recombinant viral stock, and tim e course analysis
o f recombinant protein expression.
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The calculation o f the doubling tim e for insect cells was as followed:
Ni = Noete Ni= Number o f cells at present time.
No=Number o f cells at initial time.
t= length o f time in hour
T2 (doubling time) = (In 2) / k
An adjustment period was needed to culture the insect cells after thawing. During
the adjustment period, longer doubling time, lower cell viability and lost of cell adhesion
were frequently observed. From my own experience, this adjustment period ranged from
a few days to up to 2 weeks. High-5 cells tend to have a shorter adjustment period than
Sf-9 cells, and usually two passages were sufficient for adjustment period. It would take
up to three to four passages for the insect cells to adopt to a different medium product
from other company. It is not recommended to use the insect cell line that has an
adjustment period longer than two passages, because the insect cell line has a limitation
up to 30 passages. After 30 passages the insect cell line begins to enter a crisis stage
characterized by a significant degree o f apoptosis, longer doubling time and loss o f cell
adhesion.
H igh-Five cells
High-Five insect cells were grown in Excell 400 (JRH Biosciences) serum-free
medium. High-Five cells had very sim ilar characteristics as Sf-9 cells and were used in
tim e course analysis o f recombinant protein expression.
19
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Baculoviruse Agarose for plaque assay
The final agarose solution for agarose overlay was between 0.8% to 1%. Agarose
concentration less than 0.8% did not solidify well, and concentration over 1% would
cause damage to the Sf-9 cells.
Primers used to detect the presence o f TIMP-2 cDNA within the Baculovirus
expression vector
The Baculovirus forward primer, 5,-TTTACTGTTTTCGTAACAGTnTG-3'
targeted the position -4 4 which was the upstream of the polyhedrin gene and the
baculovirus reverse primer, 5'-CAACAACGCACAGAATCTAGC-3’ targeted to position
794 located downstream o f the partially deleted polyhedrin gene. These primers were
used in the PCR analysis to confirm the presence of TIMP-2 1-101, Cysl3Ala insertion
into the pBlueBacHis 2B expression vector.
Western Blot Analysis
Concentrated culture medium and cell pellet from the infected cells were
solubilized in Laemmli Buffer (62 mM Tris-HCl, pH 6.8; 2% SDS, 25% glycerol), and
separated by 4%-20% Tris-Glycine gradient gel (Novex). Proteins were transferred to
nitrocellulose paper for an hour at 100V at 4°C. The transferred nitrocellulose was
incubated in 5% dry m ilk overnight to prevent non-specific binding. A polyclonal anti-
rTTMP-2 rabbit antibody (diluted 1:2000) and a monoclonal anti-Xpress antibody (diluted
20
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1:2500) were used as primary antibodies to recognize the recombinant protein. Anti
rabbit goat-conjugated o f alkaline phosphatase antibodies were used with the presence o f
BCIP and NBT substrates for the visual detection o f the recombinant proteins.
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RESULTS
Generation of rTIMP-2 1-101, Cysl3Ala
PCR was used to generate TIMP-2 1-101, CysI3A la cDNA. The template used in
the PCR reaction was pCDNAHMIdelta Cysl3Ala (HMI is identical to TIMP-2), that
had cysteine being replaced by alanine at the position 13 (Figure-4). The sense prim er 5 -
TCCGAGCTCGTGCA GCTGCTCCCCGGTGC-3’ had a Sac I restriction site at the 5’
end and an antisense primer 5 '-CTCC AAGCTTAAC AGAGGGGTGATGTGC ATCTI G-
3’ had a Hind m restriction site at the 3’end and a stop codon at the position 102. These
restriction sites created an overhanging end allowed the TIMP-2 1-101, Cysl3Ala cDNA
to be inserted in frame and with proper orientation into pBlueBacHis 2B expression
vector. The PCR cycles started with 5 minute at 94°C to denature the template DNA,
followed by 3 repeated cycles started at 94°C for 1 minute, 50°C for 1 minute, to allow
the hybridization o f the primers, and then 72°C for 1 minute in order for Taq polymerase
to incorporate the correct bases. 35 addition amplification cycles with 1 minute at 94°C, 1
minute at 55°C and 1 minute at 72°C were immediately followed to obtain the mutated
PCR products.
The presence o f PCR products with an expected size o f 324 base pairs was
confirmed by agarose gel electrophrosis and the product was purified by QIAquick PCR
purification kit to remove any impurities from the PCR reaction. The TIMP-2 1-101,
Cysl3A la PCR product and pBlueBacHis2B were both digested by SacI (20 U) and
HindTTT (20 U) restriction enzymes for a minimum o f 4 hours according to the
manufacturer's specification. Both restriction digested products were purified by
22
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QiAquick PCR purification kit and ligated overnight at 37°C. In the ligation reaction the
molecular ratio o f TIMP-2 1-101, Cysl3Ala PCR product over pBlueBacHis2B was
maintained at 3:1. The newly constructed plasmid pBlueBacHis2B with TTMP-2 1-101,
Cysl3A la was propagated by transformation into DH5a competent cell with heat shock
which were then selected from amplicillin-containing plates. The selected colonies were
cultured overnight and the plasmids were isolated by Qiagen Plasmid M ini kit (Figure-
4A.)
The insertion o f TIMP-2 1-101, C ysl3A la PCR product into pBlueBacHis 2B
expression vector was confirmed by loading and running 1.8 pg o f material in 1% agrose
gel to verify its size o f646 bp (containing partial polyhedrin gene and TIMP-2 1-101,
Cysl3Ala, a 324 bp PCR product.) [Figure-5B]
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S ac I
S’ - KS CGAGCT CGT GCAGCT GCI CCCCGG'I GC- J
1 2 8
3 t S n C T W 3 3 1 G W G I G G G A S A C A A I 1 C G A A C C C - 'S
U na*
PCR Reactions
5 min at
lmin at
1min at
1min at
1min at
1 min at
1 min at
10 min at
3 cycles
35 cycles
72° C
0X174
5 0X174
1353 bp
1078 bp
872 bp
603 bp
324 bp 310 bp
234 bp
Figure 4 . Generation o f T IM P -21-101 C ysl3AIa by PCR
A template pCDNAHMIdelta, in which a Cysteine was replaced by Alanine at the position 13, was used
to generate rTIMP-2 1-101 C ysl3A la in a PCR reaction using a sense prim er containing S a d restriction
site an antisense primer containing Hindm restriction site. First and last lane are $X174 DNA marker.
Lanes 1 to 5 are the replicate o f the PCR products which confirmed that a 324 bp o f TIMP-2 1-101 Cysl3AIa
cDNA product was generated
24
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ATG 6X Histidine R e g io n EK Site Multiple Cloning Site
Polyhedrin Promoter
Base: 7-94 — ^
Recombination Sequence
(ORF 1629)
B ase: 1256-456
5’ LacZ Fragment
Base: 4551-3439
Col El Region
Base: 26
Ampidllin Resistance Gene
Base: 1643-2500
Figure 5. T IM P-21-101, C ysl3A la Construction
B. The TIMP-2 delta 128 C ysl3A la mutant cDNA was used as template to replace codon 102 (GAC) by a stop
(TAA) codon, by PCR mutagenesis using a sense primer containing S a d site and an antisense primer containing
HindHI site. The PCR product was digested with both S ad and HindHI, and subdoned in frame into the
pBlueBacHis 2B expression vector. The mutant TIMP-2 was located downstream o f a polyhistidine tag sequence
and an enteroldnase recognition and cleavage site.
C. The correct insertion o f TIM P-2 1-101, Cysl3A Ia PCR product into pBlueBacHis 2B expression vector was
confirmed by loading 1.8 p g o f material and running 1% agrose g d to verify the size o f the product to be
636 bp (containing partial polyhedrin gene and TIM P-2 1-101, C ysl3A la, a 324 bp PCR product)
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Sequencing o f rTTMP-2 1-101, Cysl3Ala
The sequencing o f rTTMP-2 1-101, C ysl3A la was carried out using the Taq Track
Sequencing System (Promega) to confirm and verify the proper ligation, orientation of
pBlueBacHis2B-TTMP-2 1-101 Cysl3Ala, and the absence o f mutations created by Taq
polymerase. This sequencing system used [a-3 S S]dATP and had the advantage of
reducing the exposure time to 24 hours. Also 7-deaza-dGTP was used to replace dGTP in
the reaction to increase the resolution o f band compression.
10 pg o f pBlueBacHis2B-TIMP-2 1-101, Cysl3Ala was treated by alkali
denaturation (2 M NaOH, 2 mM EDTA, 5 M ammonium acetate pH 7.5). To increase the
resolution of the plasmid sequence, two identical sets of sequencing reactions were
prepared. The first set o f extension and labeling reaction was loaded and ran at a constant
voltage (1800V) to the half way o f the sequencing gel (about 2 hours) then the second set
o f reaction was loaded and proceeded at the same voltage. A fter the X-ray exposure, the
sequence was read and is shown Figure-6.
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Figure - 6 Sequence Confirmation
[polyhedrin foward priming site(l)]
Temp : I GATATCATGCAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAArAfyrr 72
c D N A --------------------------------------- TTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTT
[start codon (2)] [Polyhistidine Region (3)]
Temp: 73 TTGTAATAAAAACCTATAAATATGCCGCGGGTTTCTCATCATCATCATCATCATGG TATGG TTAGrA Tr, 139
cD N A TTGTAATAAAAACCTATAAATATGCCGCGGGTTTCTCATCATCATCATCATCATGGTATGGCTAGCATG
a a : m p r g s h h h h h h g m a s m
[Entero-kinase Site (4)] [guanine base(5)][Cysteine 1 (6)]
Temp 140 ACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGAGCTCGTGCAGC 206
cDNA ACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGAGCTCGTGCAGC
AA: T G G Q Q M G R D L T D D D D K D P S S CS
[Cys 13 A la mutation(7)]
Temp: 207 TGCTCCCCGGTGCACCCGCAACAGGCGTTT2££AATGCAGATGTAGTGATCAGGGCCAAAGCGGTC 273
cDNA TGCTCCC TGCACCCGCAACAGGCGTTTGCCAATGCAGATGTAGTGATCAGGGCCAAAGCGGTC
AA: C S P V H P Q Q A F A N A D V V I R A K A V
Temp 274 AGTGAGAAGGAAGTGGACTCTGGAAACGACATTTATGGCAACCCTATCAAGAGGATCCAGTATGAG 340
cDNA AGTGAGAAGGAAGTGGACTCTGGAAACGACATTTATGGCAACCCTATCAAGAGGATCCAGTATGAG
a a : S E K E V D S G N D I Y G N P I K . R I Q Y E
Temp 341 ATCAAGCAGATAAAGATGTTCAAAGGGCCTGAGAAGGATATAGAGTTTATCTACACGGCCCCCTCCTCG 410
cDNA: ATCAAGCAGATAAAGATGTTCAAAGGGCCT— G AAGG AT AT AGAGTTT ATCT AC ACGGCCCCCTCCTCG
A A : I KQI K M FK G P E f C D I E F I Y T A P S S
Temp: 411 GCAGTGTGTGGGGTCTCGCTGGACGTTGGAGGAAAGAAGGAATATCTCATTGCAGGAAAGGCCGAG 477
cDNA: g c a g t g t g t g g g g t c t c g c t g g a c g t t g g a g g a a a g a a g g a a t a t c t c a t t g c a g g a a a g g c c g a g
a a : a v c g v s l d v g g k k e y l i a g k a e
Temp 478 GGGGACGGCAAGATGCACATCACCCTCTGTGACTTCATCGTGCCCTGGGACACCCTGAGCACCACC 544
cDNA: GGGGACGGCAAGATGCACATCACCCTCTGTGACTTCATCGTGCCCTGGGACACCCTGAGCACCACC
a a : g d g k m h i t l c d f i v p w d t l s t t
27
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[Stop Codon (8)]
Temp: 545 CAGAAGAAGAGCCTGAACCACAGGTACCAGATGGOCTGCGAGTGAAGCTTGGAGTCGACTCTGCT 611
cDNA: CAGAAGAAGAGCCT CACAGGTACCAGATGGGCTGCGAGTGAAGCTTGGAGTCGACTCTGCT
a a : QKKSLN HRY QMG CE*
Tem p: 612 GAAGAGGAGGAAATTCTCCTTGAAGTTTCCCTGGTGTTCAAAGTAAAGGAGTTTGCACCAGACGCA 678
cDNA: GAAGAGGAGGAAATTCTCCTTGAAGTTTCC----------------------------------------------------------------------------------
a a :
(Temp=Template Sequence; cDNA= Confinned Sequence; AA=Amino Acid Sequence)
Figure 6. Sequence Confirmation
This is the sequence confirmation of the ligation o f TIMP-2 1-101, Cysl3Ala PCR
product into pBlueBacHis2B expression vector. This sequence map confinned: (1). The
polyhedrin foward priming site (2). ATG codon (3). 6X Histidine region (4). Entero-
kinase Site (5).The guanine base (that was added to make sure the ligation is in frame)
(6). Cysteine 1 (7). Cysl3Ala mutation (8). Stop codon after cysteine 101.
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Transfection into the Sf-9 cells
The transfection o f Bac-N-Blue DNA® linearized AcMNPV (lp g ), pBacBlueHis
2B-TIMP-2 1-101, Cysl3A la (4 |ig), and InsectionPlus® liposomes from Invitrogen (20
pi) were mixed together in a 1 ml o f Grace’s insect media and incubated at room
temperature for 15 minutes (Figure-7). The transfection mixture was then added dropwise
to a 100 mm dish that contained 2.5 X 106 Sf-9 cells and the cells were allowed to
incubate at room temperature for four hours. Rocking side-to-side was needed to make
sure all the transfection mixture spread evenly on top o f the Sf-9 cell monolayer. After
addition o f 1 ml o f TNM-FH medium, the transfection dishes were sealed and incubated
for 72 hours at 27°C.
Sf-9 insect cells were plated at 70% confluence to ensure there was enough
surface area for the liposomes to bind to the cells. If Sf-9 cells were plated too densely, it
would decrease the transfection efficiency.
There are specific changes in cell morphology that indicate a successful
transfection. These cells were carefully examined under an inverted phase microscope at
400X magnification. For the presence o f these changes the signs o f viral infection were
classified as early stage (within the first 24 hours), late stage (24-72 hours), and very late
stage (over 72 hours). At the late stage o f transfection, budded viruses released into the
medium were observed. The transfection supernatant was then assayed for its ability to
form recombinant plaques in Sf-9 cells grown agarose. (Figure-8)
29
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Circular Bac-Biue DNA
(Invitrogen)
Para •“ » ' «-*« PlKa
Cleave by Bsu361
Recombination #1
Linearized Bac-N-BIue DNA
Recombination #2
Linearized Bac-N-BIue DNA
Para P is a
pBlueBacHis2
pB(ueBacHis2
Recombination
Recombinant AcMNPV DNA
(TI MP- 2, 1 -101 , Cys13AIa)
Figure 7 . Liposom al C otnuuftction o f Sf9 insect cells.
Circular Bac-N-BIue DNA* from Invitrogen, was restriction digested by Bsu36 which eliminated part o f thepolyhedrin gene and
ORF 1629 to enhance the recombination process. During the liposomal cotransfection, with the aid o f IhsectionPlus liposomes,
Un»«riT»H Bac-N-Blue DNA was allowed to recombined with pBlueBacHis 2B- TIMP-2 1-101, C ysl3A la with two possibilities to
a recombinant AcMNPV - TIMP-2 1-101, C ysl3A la viruses. The polyhedrin promoter (Pm) was a strong promoter located
at the upctr-arr o f inserted TIMP-2 1-101,C ysl3A la cDNA for driving an efficient expression. The Pm . promoter, adjacent to Pm was
used to drive the expression o f lacZ gene, in another direction, to produce p-galactosidase.
30
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Figure 8. Stages of Transfection
The signs o f transfection were examined under an inverted phase microscope at 400X magnification. Signs o f viral
infection were classified as early, within the first 24 hours (Figure A); late stage, 24-72 hours (Figure B, arrows 1
and 2 shown that the cells were swollen and increased in diameter with granular appearance); and very late stage,
over 72 hours (Figure C, arrows 3 and 4 indicated that the cells began to lysis.) At the very late stage o f transfection,
budded viruses were released into the medium (Figure C, arrow 5).
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Table #5 The Observations of the Stages of Transfection
Early Stage o f Transfection {within 24 hours')
1. Cell diameter increased. Some o f the Sf-9 cells had swollen about 25%-50% in
diameter.
2. The size o f the cell nuclei increased. Swollen nuclei were observed in transfected Sf-9
cells.
Late Stage o f Transfection (within 24-72 hours)
1. Sf-9 cells appeared to slow their growth compared with the normal Sf-9 cells.
2. Detachment from the plate occurred.
3. Granular appearance observed, which indicated the sign o f viral budding.
4. Sf-9 cells began to lysis, which account to about 25% o f the population.
5. Low viability (from 47% to 65%).
Very Late Stage o f Transfection (After 72 hours)
Cell lysis and low viability: About 85% o f Sf-9 cells lysized and fragmented. Viability of
Sf-9 cells were below 30%.
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Plaque Assay
72 hours after transfection, the supernatant from the transfected dishes should
have about 80% recombinant virus. It was necessary to separate the recombinant virus
from the non-recombinant AcMNPV, other unwanted recombinant virus, and
contaminants. Since the non-recombinant virus can propagate and infect more efficiently
than the recombinant virus, the plaque assay was used to select out the recombinant virus
and also to determine its titer.
150 pi o f 50 mg/ml X-gal was added into TNM-FH medium. The Sf-9 cells were
seeded at a density o f 5 X 106 cells at 100 mm plates with a viability o f 98%. The plate
was rocked for 10 minutes to ensure that Sf-9 cells were distributed evenly. 10-fold serial
dilutions o f the transfection viral stock from 10'1 to 10'3 were prepared. The diluted
recombinant virus from transfection viral stock was added dropwise to Sf-9 cell plates
and then allowed to incubate for one hour on a rocking platform. Immediately after
incubation, agarose-TNM-FH medium solution containing X-gal was overlaid on top o f
the plates. The plates were then sealed for six days until the blue plaques formed.
Titer Calculation:
(1/dilution) X (number o f plaques) = plaque forming unit (pfu)/ml
T able #6 P -l Recom binant V iral Stock T iter
Viral Dilution Blue plaques observed Titer (pfu/ml)
1 0 * 2 8 0.8 X 10J
10'3 3 3.0 X 10J
The P -l viral recombinant viral stock was generated by seeding 5X 105 Sf-9 cells
in a multi-well plate with 2 ml o f TNM-FH medium in each well. A pasteur pipette was
33
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used to transfer the blue plaque agarose plugs to infect the Sf-9 cells in m icrotiter plate
wells. M icrotiter plate was sealed and incubated for 3 days. The supernatant in the wells
o f microtiter plate was designated as the P-l recombinant viral stock.
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PC R Analysis of Recom binant Baculovirus
The PCR technique was used to confirm the isolation of pure recombinant
plaques. It was a safe, quick, and nonradioactive method to identify the presence o f
TIMP-2 1-101, Cysl3A la cDNA and the partial polyhedrin region o f the baculovirus.
The P-l recombinant viral stock after 3 days incubation was concentrated in 0.75
ml o f 1 M NaCl with 20% PEG with centrifugation at 13,000 rpm for 10 minute. The
precipitate was then incubated with Proteinase-K (10 mg/ml) at 50°C for one hour to
destroy the insect cells and release its DNA. A phenokchloroform extraction method was
performed to extract the DNA which was allowed to precipitate in the presence o f 3 M
sodium acetate, 5 pi o f glycogen (2 mg/ml), and 100% ethanol. The DNA pellet was
washed with 80% ethanol and traces o f ethanol were removed by evaporation before the
pellet was resuspended in sterile water. Two PCR reactions were carried out. The first
PCR reaction used the primers that generated TIMP-2 1-101, Cysl3Ala to detect the
presence o f mutant TIMP-2 cDNA (324 bp). The PCR reaction used the baculovirus
forward and reverse primers (lOOng/pl) to detect the presence o f partial polyhedrin gene
in addition to the TIMP-2 cDNA (636 bp). Both PCR reactions used 5 pi o f viral DNA,
5pl o f 10X PCR Buffer, lp l of 25 mM dNTPs, and 1.5 units of Taq Polymerase. PCR
cycles were run according to the protocol described at the section o f materials and
methods for the generation of TIMP 1-101, Cysl3A la cDNA.
From the 1% agarose gel electrophoresis the expected 324 base-pair (lanes 3 & 4,
with primers 3 and 4) and 636 base-pair PCR products (lanes 6 & 7, with primers 1 and
2) were detected (Figure- 9).
35
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PC R ANALYSIS O F RECOMBINANT VIRUS
Baculovirus Reverse PCR
Priming Site
Enterokinase Region
Baculovir Toward PCR Polytiistidine Region
Priming Site . | —
TIMP-2 (1-101. Cys13Ala)
EK Cleavage Site
Primer 3 Primer 4
Primer 1
Primer 2
600 bp-
500 bp-
400 bp-
300 bp-
-636 bp
-324 bp
Figure 9. PCR Analysis o f Recombinant Virus
The PCR analysis was used to detect the presence o f the recombinant DNA clone. Lane 1
is 100 base-pair standard DNA marker. Lane 3 and 4 are the PCR product generated in
the presence o f primer 3 and 4, detecting the presence o f TIMP-2 1-101 Cysl3A la. Lane
6 and 7 used baculovirus foward and reverse primers 1 and 2 to detect the presence of
partial polyhedrin gene.
36
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G eneration o f H igh-Titer Recom binant V iral Stocks
The P -1 recombinant viral stock had a low titer o f 3 X 103 pfu/ml. In order to
perform expression studies, 1 X 108 (pfu/ml) was required, therefore it was necessary to
generate a high titer recombinant viral stock.
2 X 106 log phase Sf-9 cells were seeded in 25 cm2 flask with 5 m l TNM-FH. P-l
recombinant viral stock (20 pi) was added to the 25 cm2 flask, which was incubated at
27°C for 7 days to allow a total lysis o f the Sf-9 cells. The viability o f Sf-9 cell was
determined by the trypan blue exclusion analysis. The complete lysed supernatant was
designated the P-2 recombinant viral stock. One ml aliquot o f this P-2 recombinant viral
stock was preserved at -80°C for long-term storage. The remaining P-2 recombinant viral
stock (4 ml) was added to a 250 ml suspension o f log-phase Sf-9 cells w ith a density of
2.0 X 106 cells/ml with constant stirring at 65 rpm for 7 days at 27°C in a spinner flask.
Pluronic F-68 (0.1%), a surfactant that decreases cell membrane shearing, was
added to the culture medium to prevent the cell shearing. The supernatant in this flask
was designated as P-3 recombinant viral stock and the titer o f this viral recombinant stock
was determined, 2 X 108 pfu/ml with neutral red overlay [1 % Neutral Red Stock (10
mg/ml)] to enhance the visibility o f blue plaques.
Table # 7 P-3 Recom binant V iral Stock T iter
Viral Dilution Blue plaques observed Titer (pfu/ m l)
1 0-* 164 1.64 X 10“
10’7 19 1.9 X 108
10'5 2 2.0 X 108
37
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Time Coarse Expression
Since each recombinant virus would have different kinetics in term of its infection
and expression, a time course analysis o f the expression o f the recombinant protein was
needed to indicate the optimal time for the recombinant protein expression.
To comprehend the kinetics o f the recombinant virus, has a titer o f 2.0 X 108
pfu/ml, different sets o f multiplicity o f infection (MOI) were tested for High-Five insect
cells (6.18 X 107 cells, 95% viable). Volume of inoculum needed for recombinant protein
expression was calculated in the following formula:
Inoculum needed (ml) = [ MOI (pfii/cell) X number of insect cells]/ titer of virus (pfu/ml)
Different sets o f time course experiment samples were prepared (Table #6.) The infection
process was performed in 250 ml spinner flask with constant stirring o f 60 rpm.
Table #8 Time Coarse Expression with Different MOI by Infecting High-Five cells
Set A Set B Set C
MOI 3 6 9
Volume o f recombinant
viral stock
1.85 ml 3.71 ml 5.56 ml
Total High-Five cells 6.18 X 107 cells 6.18 X 1 0 'cells 6.18 X 107 cells
Volume o f Excell 400 93 ml 91 ml 85.5 ml
Total Volume 125 ml 125 ml 125 ml
Every 12 hour, 1 ml o f aliquots was removed and transferred into the eppendorf
tubes, and centrifuged down (5,000 rpm, 10 min.) to separate pellet and supernatant. Cell
viability was monitored by trypan blue exclusion assay. (Figure 9B & 10B)
38
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Each pellet from different time point, was resuspend in 100 pi o f sodium
phosphate buffer (20 mM, pH 7.0) and treated with repeat freezing in liquid nitrogen and
thawing at 37°C to break up the cell wall. The cell debris was removed by centrifugation
o f 13000 rpm for 10 minutes. Cocktail o f protease inhibitors was added to the pellet and
supernatant samples and resuspended in Laemmli Buffer. 20 pi o f samples and 10 pi o f
Laemmli Buffer were boiled for 10 minutes and loaded into the wells o f 4%-20% Novex
Tris-Glycine gradient gels to separated proteins according to size. The gradient gel was
stained with coomassie blue to detect for the presence o f the recombinant protein.
Western blot analysis with Anti-Xpress monoclonal antibody (against enterokinase
region) and Goat-anti-TIMP-2 antibody were used to detect the presence o f the
recombinant protein. (Figure 8A) The molecular weight o f the recombinant protein
(expected to be 15 kDa) was confirmed by using SeeBlue marker (Novex) as standards
(Figure 10C & 11 A.)
To prevent the proteolytic degradation o f the recombinant protein, a mixture o f
protease inhibitors, containing Pepstain A (lpg/m l), Leupeptin (5 pg/ml), PMSF (10
pg/ml) and Aprotinin (5 pg/ml) was added immediately after the supematent was
collected.
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M B
1 2 U .
Pellet
Supernatent —
Anti-Xpress
Antibody
*
- r - . « ■ *
0 12 24 36 48 60 72 84
Pellet
Supernatent — <
Anti-TIMP-2
Antibody
The Viability of Infected of Sf-9 Cells V- 2-B214- 2-3302X F V 2 .0 .5 7 T
LogfKDa]
Time (Hours)
Figure 10 - Time Course Analysis o f TIMP-2 1-101. C ysl3A la expression in Sf-9 cells
B. Western Blot Analysis o f aliquots from the culture medium and cell pellet o f Sf-9 cell infected by recombinant
TIMP-2 1-101 C ysl3A la virus (MOI=6). B oth polyclonal anti-rTIMP-2 rabbit antibody and Anti-Xpress antibody
(Invitrogen) were used to detect the presence o f mutant iTIM P-2 expression over time:
B. Infected Sf-9 cells viability over time was determined by cell counting and trypan blue exclusion.
Molecular weight determination o f the bands (R f value o f 0.72) detected by Western blot analysis using SeeBlue
m arker (Novex) as the standard. The R f value indicated a molecular weight o f IS Kda, consistent with the predicted
molecular weight o f the recombinant TIM P-2 1-101, C ysl3A la protein.
40
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012 24 36 48 60 72
2 5 0 k O a-
98kD a-
6 4 k D a-
50kO a-
36kD a-
30kD a-
16kD a-
6kD a-
p p PPf
15kDa
B
The Viability of Infected Hlgh-Fhw Calls
Western Blot Analysis on collected supernatent samples
1 2 3 4 5 6 7
*-*. -- ^ jg g p .— 250kDa
98k0a
' — - ■ * g p <— 64k0a
— 50k0a
36k0a
OkDa
16KDa
6kDa
15kDa-
Figure 1 1 - Time Course Analysis o f TIMP-2 1-101 Cysl3Ala expression in High-5 cells
B. Western blot time course analysis of aliquots from the culture medium of High-Five
insect cells infected by recombinant TIMP-2 1-101 Cysl3Ala (MOI=6). Polyclonal anti-
rTIMP-2 rabbit antiserum was used to detect for the presence of mutant rTIMP-2 expression
over time. Lane 1 is the SeeBlue marker. Lane 2 is the uninfected High-Five sample. Lanes 3
to 8 are the time course samples.
B. The viability of infected High-Five insect cells
The viability of infected High-Five insect cells with various sets of MOL was determined by
cell counting and trypan blue exclusion.
C. Western Blot analysis on collected supernatant samples.
Lane 1 and 7 are SeeBlue Marker. Lane 2 and 3 are 48 hour post-infection supernatant with
MOI of 6 and 9 respectively. Lane 4,5, and 6 are 72 hour post-infection supernatant with MOI
of 3,6, and 9 respectively. .
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DISCUSSION
It was reported that the expression o f recombinant mouse TTMP-1 by bacterial
system using E. Coli, had a very low yield o f active protein (about 0.5%) and most
recombinant proteins had resulted in inclusion bodies (42). The mammalian system was
previously used to express and purify the recombinant TIMP-2 from the conditioned
medium o f dihydrofolate reductase-deficient CHO cells transfected with vector pDSRa2
containing human TIMP-2 cDNA (27). Although the mammalian system worked
successfully, but the process is time consuming, therefore we are interested in generating
active TIMP-2 proteins at a higher yield and at a faster pace.
Recently the baculovirus expression system was sought as an ideal method of
expressing active protein. In fact, mouse TIMP-1 was expressed by baculovirus
expression system, in which the mouse TIMP-1 cDNA was placed under the control o f
the strong polyhedrin promoter in pBlueBac II transfer vector. The recombinant virus
with TIMP-1 cDNA was used to infect Sf-9 cells and about 3 mg o f glycosylated active
mouse recombinant TIMP-1 was obtained from one liter of conditioned medium (43).
The baculovirus expression system produced about 200-fold higher expression than the
previous attempted bacterial system (43). Major determinants such as glycosylation,
successful folding, and the partial elimination o f G-C rich 5’ noncoding sequences, a
region that has an adverse effect on translation (44,45) in recombinant transfer vector, are
all factors that contribute to the success o f baculovirus expression system.
Also TTMP-4, a new member o f the TIMPs was obtained by 5-liter Bioreactor in a
baculovirus expression system. When harvested at 70 hours post-infection, it produced a
42
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yield o f approximately 1.7 mg/ 2X107 cells. A 4-step chromatography procedure that
included a strong cation exchange chromatography, a weak cation exchange
chromatography, a hydrophobic interaction chromatography, and a size-exclusion column
was used to purify the protein (46). A step elution by using different concentrations o f
NaCl in strong cation-exchange column was the first approach to purify TIMP-4. The
fractions were checked for anti-MMP activity by using gelatin degradation assay and the
active fractions were pooled. A weak cation-exchange column chromatography followed
by the hydrophobic interaction chromatography was used to further purify and
concentrate the active fractions. The size-exculsion column using Superdex S-200 was
used as the final step o f the purification to select out TIMP-4 based on the molecular
weight.
In this report, we have started at a smaller scale o f protein expression (250 ml),
and one-step nickel affinity chromatography to try to purify the protein by using the
specific binding o f the 6X histidine tag to the nickel column. Smaller scale (250 ml)
production o f recombinant TIMP-2 1-101, Cysl3A la was generated by infecting both Sf-
9 and High-Five insect cells. Unfortunately, the expression was low and the protein could
not be detected by SDS-PAGE with coomassie blue staining, although the presence o f the
recombinant protein was confirmed by Western blot analysis using both an anti-Xpress
and an anti-TIMP-2 rabbit antibody. Purification o f recombinant protein is currently in
progress. Difficulty in purification using nickel affinity chromatography to isolate the
recombinant proteins produced by the infected Sf-9 cells, was due to the presence o f
serum and lower pH value (pH 6.22) in the TNM-FH. Lower pH resulted in the
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
deprotonating o f the histidine residue and therefore lost the binding affinity to the nickel
affinity column.
The purification o f recombinant proteins from cell pellet and supernatant samples
o f infected High-Five cells, which would eliminated the serum problem, are also
currently pursued. Hopefully after the purification o f the TIMP-2 1-101, Cys 13 Ala
protein generated by the baculovirus expression system with nickel affinity
chromatography, the yield w ill be sufficient to allow to proceed with functional studies.
One o f the multi-function of TIMP-2 is its ability to stimulate growth. It was
demonstrated that in the presence of at least 4 ng/ml o f TIMP-1 or 0.1 ng/ml o f TIMP-2,
resulted in an increase o f the incorporation o f [3 H]-thymidine in Raji cells, a Burkitt
lymphoma cell line (47). The presence o f 10 ng/ml o f TIMP-2 has a maximal effect on
the growth o f Raji cells, demonstrated by a 4-fold increase in the DNA content and also a
2-fold increase in DNA content for human gingival fibroblast (Gin-1) (57). When TEMP-
2 1-101, Cysl3A la is purified, it will be tested for its ability to stimulate the growth o f
Raji and Gin-1 cells using [3 H]-thymidine incorporation. By comparing the full-length
TIMP-2 with TIMP-2 1-101, Cysl3Ala, one could determine if the first two loops o f N-
terminal domain are responsible for the growing and stimulating effect.
The binding capacity o f TIMP-2 1-101, Cysl3AJa to the cell surface can be
studied by Scatchard analysis using iodinated TIMP-2 in the presence o f human HT1080
cells and Raji cells (27). The binding o f 1 2 5 I-TIMP-2 to Raji cells was examined and
analyzed using Scatchard plot by Hayakawa et al., who suggested two types o f receptors
with high affinity (Kd=0.15 nM) and low affinity (Kd=35 nM) were presented (57). The
specificity o f the binding is determined by competition in the presence o f 100 fold excess
44
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o f unlabeled rTIMP-2. It was demonstrated that TIMP-2 l-128,C ysl3A la lost its high
affinity binding by two order o f magnitude upon the delection o f the C-terminal domain
(27). I would predict that TIMP-2 1-101,Cysl3Ala will have a lower binding affinity,
because the third loop o f the N-terminal domain was deleted.
The antimetalloproteinase activity o f TIMP-2 1-101, Cysl3Ala can be measured
by two different assays, collagenase inhibition assay and gelatin reverse zymography. In
collagenase inhibition assay, TTMP-2 1-101,Cysl3Ala will be added to a well coated with
[I4 C]-labeled rat skin type-I collagen in the presence o f APMA activated crude
collagenase. The antimetalloproteinase ability o f TIMP-2 l-101,Cysl3A la can be
measured by counting the amount of radioactivity release from [I4 C]-labeled rat skin
type-I collagen into the supernatant. From this experiment an IC50 (inhibition
concentration that reached to 50% inhibition o f MMP activity) for TIMP-2 1-
101,Cysl3Ala can be determined in the presence of a standard amount o f MMPs with an
increased amount o f TIMP-2 l-101,Cysl3Ala.
Gelatin reverse zymography is a faster screening method to detect the presence of
antimetalloproteinase activity. In this assay the gelatin and gelatinase are polymerized in
SDS-PAGE gel, and TIMP-2 1-101, Cysl3Ala is loaded and electrophoresised as a
regular SDS-PAGE gel. The gelatinase is then activated at the presence o f CaCh at 37°C.
An inhibition zone found on the reverse zymography gel, would suggest that TIMP-2 1-
101, Cysl3Ala has retained its antimetalloproteiase activity.
The generation o f full length TIMP-2 and TIMP-2 1-128, Cysl3A la by
baculovirus expression system is currently undertaken. TIMP-2 1-128, Cysl3AIa w ill
serve as a control to show that the baculovirus expression system is suitable for the
45
permission of the copyright owner. Further reproduction prohibited without permission.
expression system o f the TTMP proteins. There is a possibility that the presence o f four
extra amino acids presented before the first Cys residue of the mature protein may have
an effect on the formation of disulfide bonds and the functional activity o f the mutant
TIMP-2.
If full length TIMP-2 was expressed by baculovirus expression system and was
non-functional, it would suggest that the presence o f additional amino acids at the N-
terminal end o f TIMP-2 substantially affect the tertiary structure o f the protein.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Creator
Kwan, Stephen Triumph-Shien
(author)
Core Title
Generation of mutant tissue inhibitor of metalloproteinases-2 (TIMP-2) in the baculovirus expression system
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Graduate School
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Master of Science
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Biochemistry and Molecular Biology
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biology, cell,biology, molecular,OAI-PMH Harvest
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English
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DeClerck, Yves (
committee chair
), [illegible] (
committee member
), Tokes, Zoltan A. (
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