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Preparation and characterization of Tf-G-CSF fusion protein

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Preparation and characterization of Tf-G-CSF fusion protein

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PREPARATION AND CHARACTERIZATION OF Tf-G-CSF FUSION PROTEIN

by

Hsin-Fang Lee

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2008

Copyright 2008 Hsin-Fang Lee

ii

ACKNOWLEDGMENTS
My appreciation goes to Dr. Wei-Chiang Shen for his guidance and
encouragement during my graduate work. I am grateful for the opportunity to join
his laboratory.
My appreciation also goes to my committee members, Dr. Curtis Okamoto and
Dr. Clay Wang for their advice in my work and thesis.
I would also like to thank Mrs. Daisy Shen for her valuable support in both
technical and emotional aspect.
Additionally, I would like to express my thankfulness to my labmates for their
support and friendship: Xiaoying Chen, Robert Mo, Nurmamet Amet, Yan Wang,
Jennica Zaro, Randall Kenien, Ling Ren and Likun Fei.
Finally, I would like to thank my family and my friends for their support.
They are always there for me during bad time and good time.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii
LIST OF FIGURES v
ABBREVIATIONS vi
ABSTRACT vii
INTRODUTION
1. Protein drugs xxxxxxxxxxx 1
2. Transferrin and transferring receptor ). 1
2.1 General information of transferrin and transferrin receptor . 1
2.2 Application of transferrin in oral delivery of protein drugs ). 3
3. Granulocyte colony stimulating factor (G-CSF) 4
4. G-CSF-Tf fusion proteins -) 4
5. Specific aims ) .... 5

MATERIALS AND METHODS
1. Cell culture B 7
1.1 HEK-293 cell .. 7
1.2 NFS-60 cell 7
1.3 Caco-2 cell 8
2. Plasmid transformation 8
3. Plasmid purification / 9
3.1 Small scale plasmid purification 9
3.2 Large scale plasmid purification 9
4. PCR B 10
4.1 Generation of DNA fragment for cloning B) 10
4.2 Colony PCR B 10
5. Restriction enzyme digestion ) 11
6. DNA electrophoresis 11
7. Recovery of DNA fragments from agarose gel B 12
8. DNA ligation 12
9. Protein expression 12
9.1Small scale protein expression 12
9.2 Large scale protein expression B 13
10. SDS-PAGE ) 13
11. Western blot 14
12. In vitro activity assay 15
13. Iodination of Tf 15
iv
14. TfR binding assay xxx x xxxxx xxx x 16
15. Animal study xxx B 16

RESULTS
1. Plasmid construction B.
.l
18
1.1 Construction of pcDNA3.1-sTf-G-CSF plasmid ) 18
1.2 Construction of pcDNA3.1-sTf-H4
n
-G-CSF (n=1~3) Plasmid ). 18
2. Expression and identification of sTf-H4
n
-G-CSF (n=0~3) fusion proteins . 22
3. H4 linker increases the expression level of Tf-G-CSF fusion proteins - 25
4. Tf-H4
n
-G-CSF (n=0~2), and G-CSF-H4
n
-Tf (n=0~3) fusion proteins
exhibit similar in vitro activities ) 27
5. Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf fusion proteins shows similar
binding abilities to Tf receptor B 31
6. Tf-H42-G-CSF shows myelopoietic activity in in vivo study B 34

DISCUSSION
1. The insertion of helical linker, H4, increases the expression level of
Tf-G-CSF fusion protein . 38
2. The order of transferrin and G-CSF in fusion protein does not have
much influence on the binding of the proteins to transferrin receptor . 39
3. The in vivo efficacy of the fusion protein does not correlate well with
the in vitro activity . 40

REFERENCES . 42

v
LIST OF FIGURES
Figure 1: The schematic diagram of Tf-H4
n
-G-CSF fusion proteins. 20
Figure 2: Schematic drawing of Tf-H4
n
-G-CSF fusion proteins-expressing
plasmids. 21

Figure 3: Identification of Tf-H4n-G-CSF fusion proteins by antibody
against human transferrin. 23

Figure 4: Identification of Tf-H4n-G-CSF fusion proteins by antibody
against human G-CSF. 24

Figure 5: The comparison of the expression level of Tf-H4
n
-G-CSF fusion
proteins (n=0~3). 26

Figure 6: Evaluation of the in vitro activities of Tf-H4n-G-CSF fusion
proteins (n=0~3). 29

Figure 7: Comparison of the in vitro activities of Tf-G-CSF, Tf-H42-G-CSF,
G-CSF-Tf, and G-CSF-H4(2)-Tf fusion proteins. 30

Figure 8: Comparison of the TfR binding abilities of Tf-H4
2
-G-CSF and
G-CSF-H4(2)-Tf fusion proteins. 33

Figure 9: Evaluation of the percentage of neutrophils in white blood cells is
determined by flow cytometry. 36

Figure 10: Comparison of the in vivo activities of Tf-H42-G-CSF and
G-CSF-H4(2)-Tf fusion proteins. 37

vi
ABBREVIATIONS
Tf: transferring
TfR: transferring receptor
G-CSF: granulocyte colony stimulating factor
125
I-Tf:
125
I-labeled transferrin
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

vii
ABSTRACT
In this study, Tf-H4
2
-G-CSF fusion protein was engineered for investigating
the effects of H4 helical linker’s on the fusion protein expression and the sequence of
transferrin and G-CSF moieties on fusion protein activity. Results from this study
indicated that H4 linker not only elevated the expression but also increased the in
vitro activity of Tf-G-CSF fusion protein. Tf-H4
2
-G-CSF was also compared with G-
CSF-H4(2)-Tf for in vitro TfR binding ability, and in vitro and in vivo activity. The
results demonstrated that the change in sequence of transferrin and G-CSF moieties
had no effect on the in vitro activity and in vitro TfR binding ability of the fusion
protein. However, when administered subcutaneously to BDF1 mice, the fusion
protein with transferrin at the N-terminal domain exhibited significantly higher in
vivo myelopoietic efficacy than the fusion protein with transferrin at the C-terminal
domain.

1
INTRODUCTION
1. Protein drugs
Recombinant proteins have become an important class of pharmaceuticals over
the past decade. The protein therapeutic market is forecasted to rise at least 10% a
year. Most of the protein drugs are limited to invasive administration routes, such as
intravenous (i.v.) and subcutaneous (s.c.) injection. Frequent injection not only
causes inconvenience but also elicits adverse effect to patients. Therefore,
noninvasive delivery methods, especially oral administration, are more desirable
ways for the delivery of protein drugs. However, the development of oral delivery
system for protein drugs is a great challenge to pharmaceutical industry. The main
obstacles to oral delivery of proteins are pre-systemic enzymatic degradation and
poor penetration of the intestinal mucosa.(1) One of the approaches for the oral
delivery of protein drugs is using receptor-binding ligands as carriers to target
receptors, followed by the transcellular transport of protein drugs across the
intestinal epithelia, a process called “receptor-mediated transcytosis.(2)

2. Transferrin and transferrin receptor.
2.1 General information of transferrin and transferrin receptor.
Transferrins are the major iron-transport and regulation proteins in vertebrates
and in certain invertebrates.(3) There are 3 sub-classes of transferrins: serum
transferrin, lactoferrin, and ovotransferrin.(4) In this thesis, I will only focus on
serum transferrin.
2
Transferrin is a glycoprotein of about 80 kD. It consists of a single polypeptide
chain of around 700 amino acid residues.(3) It is synthesized predominantly by
hepatocytes. The in vivo half-life of transferrin in human is 8 days.(5) Transferrin is
responsible for binding and transporting non-heme iron from absorption sites
through blood, lymph fluid and many other biological fluids to storage or utilization
sites.(6) Transferrin is also found to exhibit antimicrobial activity. It sequesters free
iron from bacteria, thus suppresses the growth of bacteria and reduces infection.
Transferrin contains 2 domains, which are called N lobe and C lobe.(4) The
primary transferrin receptor binding site is on the C lobe, and N lobe is found to
strengthen the interaction between transferrin and target cells.(7, 8) Each lobe could
bind 1 Fe
3+
atom, and the affinities of the two lobes for Fe
3+
are similar.(9) There are
several factors involves in the binding and release of Fe
3+
by transferrin, such as
temperature, pH, chelator, and ion concentration. The presence of bicarbonate anions
is crucial for Fe
3+
binding to transferrin.(10-12) Bicarbonate anion would stabilize
the iron-binding sites, and allow Fe
3+
to coordinate tightly with amino acids which
compose the binding pockets. Furthermore, transferrin could not release Fe
3+
properly in the absence of bicarbonate anion.(13, 14) Chloride also involves in
modulating the release of Fe
3+
: chloride anion retards Fe
3+
release at neutral pH, but
accelerates the reaction at acidic pH.(15)
Transferrin receptor is a homodimer. Each monomer is about 90 kD.(16)
Binding of Fe
3+
to transferrin dramatically alters the binding affinity of transferrin for
transferrin receptor. The receptor binding affinity increases with the increasing of
3
iron content of transferrin. Diferric transferrin exhibits approximately 100 times
higher binding affinity for the receptor than apotransferrin.(9)
The binding of Fe
3+
-loaded transferrin to the receptor on cell surface is followed
by internalization, and the transferrin-transferrin receptor complex is transported to
endosomes. Then, the ATP-dependent proton pumps elevate the H
+
level in
endosomes and the iron is released in the acidic environment.(17) Apotransferrin
remains bound to the receptor in the endosome. The complex is then transported
back to the cell surface, where apotransferrin will be released.(18) The released
apotransferrin is ready for the next cycle of iron uptake. One transferrin molecule is
estimated to be able to participate the iron transport cycle as many as 100 times.(19)

2.2 Application of transferrin in oral delivery of protein drugs.
Since transferrin is a natural protein for transporting iron to cells, and there are
abundant transferrin receptors in human gastrointestinal epithelium, transferrin is
considered as an ideal carrier for oral delivery of protein drugs. In fact, previous
studies in our laboratory successfully demonstrated that therapeutic effect could be
observed in mice or rats when transferrin is used as the carrier to administer drugs
orally. Both oral administration of transferrin chemical conjugates (insulin-Tf and G-
CSF-Tf) and transferrin fusion protein (G-CSF-Tf) showed therapeutic effects.(20-22)

4
3. Granulocyte colony stimulating factor (G-CSF)
Granulocyte colony stimulating factor, also known as colony-stimulating factor
3, is a hematopoietic growth factor. G-CSF stimulates the survival, proliferation, and
differentiation of neutrophil precursors.(23, 24) G-CSF is a glycoprotein containing
174 amino acids with molecular weight around 20 kD. G-CSF is mainly produced by
macrophage. The central role of G-CSF is to control neutrophil development.
Recombinant G-CSF is used in the treatment of neutropenia which is a common
adverse effect of cancer chemotherapy or bone marrow transplantation.(25, 26)
Because of the short half-life of G-CSF (only several hours), the drug has to be
injected at least once a day.(24) Frequent injection of the drug not only causes
inconvenience and discomfort, but also induces side-effects in patients, such as
potential vein collapse. Either extending the half-life or developing orally
administrated G-CSF would provide great benefit to patients receiving G-CSF
treatment.

4. G-CSF-Tf fusion proteins
In previous work, our laboratory generated G-CSF-Tf fusion protein which was
shown to elicit the increase of absolute neutrophil counts (ANC) when orally
administered to BDF1 mice.(22) Furthermore, in order to improve the efficacy of the
fusion protein, different linkers were inserted into the fusion protein to increase the
distance between G-CSF and transferrin moieties.(27) By doing so, the steric
interference between the two moieties is expected to be minimized, and the binding
5
ability of the fusion protein to G-CSF receptor and transferrin receptors would be
increased.
Among the fusion proteins with different linkers, the one bearing 2 copies of H4
helical linker (G-CSF-H4(2)-Tf) has been shown to reveal the highest oral
bioavailability and myelopoietic efficacy in BDF1 mice.(27) Although G-CSF-
H4(2)-Tf shows higher biological activity, there is no improvement in its transferrin
receptor binding ability. This result implies that the low transferrin receptor binding
ability may not be due to the steric interference between G-CSF and transferrin
moieties.
Another possible explanation for the low receptor binding ability of the fusion
protein is that the extra peptide extending from the amino- terminal region (N-
terminus) of transferrin may decrease the binding affinity to the receptor. This
assumption could be tested by comparing the transferrin receptor binding abilities of
Tf-G-CSF and G-CSF-Tf fusion proteins. Unfortunately, the transferrin receptor
binding ability of Tf-G-CSF could not be determined because of the low yield of the
fusion protein.(27)

5. Specific aims
Previous study shows that G-CSF-Tf fusion proteins with H4 linker not only
reveal higher activity, but also have higher level of expression than fusion proteins
without H4 linker. It implies that H4 linker may increase the expression of fusion
proteins. In this study, our goal was to generate Tf-G-CSF fusion proteins with H4
6
linker to investigate whether the linker could really boost the protein production. If
we could obtain enough Tf-G-CSF fusion protein, we would compare Tf-G-CSF
with G-CSF-Tf in several aspects including transferrin receptor binding ability, in
vitro activity, and in vivo activity. The comparison would provide us with
information for further improving the bioavailability and efficacy of protein drugs
utilizing transferrin as a transport carrier.

7
MATERIALS AND METHODS
1. Cell culture
1.1 HEK-293 cells
HEK-293 cell line is derived from human embryonic kidney and obtained from
American Type Culture Collection. This cell line is cultured in DMEM medium
supplemented with 10% FBS, 0.1 mM nonessential amino acids, 2 mM L-glutamine,
50 U/ml penicillin (sodium salt), and 50 μg/ml streptomycin sulfate at 37℃ in an
atmosphere of 5% CO
2
.
For fusion protein expression, GIBCO® FreeStyle
TM
293 Expression Medium
was used to feed the cells. This medium, a chemical defined protein free medium,
was developed for the growth and transfection of FreeStyle 293-F cells (derived
from HEK-293 cells) under suspension type culture condition. In our study, the
medium was found to be able to keep HEK-293 cells in adhesive culture.

1.2 NFS-60 cells
NFS-60 is a murine myeloblastic cell line established from leukemia cells
obtained from Cas-Br-M murine leukemia virus infected mice. This cell line is IL-
3/G-CSF-dependent; therefore, it is used to measure G-CSF activity of our fusion
proteins.
NFS-60 cell line was cultured in RPMI 1640 medium supplemented with 10%
FBS, 0.1 ng/ml IL-3, 50 U/ml penicillin (sodium salt), and 50 μg/ml streptomycin
sulfate at 37℃ in an atmosphere of 5% CO
2
. RPMI 1640 medium containing only
8
10% FBS but not IL-3 was used in the measurement of the G-CSF activity of the
fusion proteins.

1.3 Caco-2 cells
Caco-2 is an intestinal cell line derived from colorectal adenocarcinoma. This
cell line has been shown to differentiate after reaching confluence. Caco-2 cell model
is commonly used to determine the GI absorption potentials of drugs. This cell line
was cultured in DMEM medium supplemented with 20% FBS, 0.1 mM nonessential
amino acids, 50 U/ml penicillin (sodium salt), and 50 μg/ml streptomycin sulfate at
37℃ in an atmosphere of 5% CO
2
.

2. Plasmid transformation
Plasmids were transformed into E. coli DH5 α competent cells (Zymo Research).
Before transformation, LB agar plates containing appropriate selective antibiotic (50
μg/ml penicillin) were pre-warmed. Mix 1~5 μl plasmid DNA with thawed
competent cells on ice, and incubate the mixture on ice for 5 min. The mixture was
spreaded onto a pre-warmed plate, and the plate was incubated at 37℃ for the
colonies to grow.

9
3. Plasmid purification
3.1 Small scale plasmid purification
QIAprep spin Miniprep kit (QIAGEN) was used for small scale plasmid
purification. Bacteria bearing plasmids were inoculated in LB medium containing
antibiotic the day before plasmid purification. After 12~16 hr of incubation at 37℃
with vigorous shaking, the bacteria were harvested by centrifugation at 7000 rpm for
10 min at room temperature. The plasmids were then extracted and purified as
described in the handbook provided by manufacturer.

3.2 Large scale plasmid purification
EndoFree plasmid mega kit (QIAGEN) was used for large scale plasmid
purification. Bacteria bearing plasmids were inoculated in LB medium containing
antibiotic and incubated at 37℃ for 12~16 hr to the make starter culture. The day
before plasmid purification, 500 μl starter culture was added to 500 ml of LB
medium and. After 12~16 hr of incubation at 37℃ with vigorous shaking, the
bacteria were harvested by centrifugation at 7000 rpm for 15 min at 4℃. The
plasmids were then purified following the instructions provided by the manufacturer.

10
4. PCR
4.1 Generation of DNA fragment for cloning
Platinum
®
Pfx DNA polymerase (Invitrogen) was used to generate DNA
fragment for plasmid construction. The condition of the reaction was as followed:
Template: 1 μl
Forward primer (10 μM): 1.5 μl
Reverse primer (10 μM): 1.5 μl
10× Pfx amplification buffer: 5 μl
10 mM dNTP mixture: 1.5 μl
50 mM MgSO4: 1 μl
Pfx DNA polymerase: 1 μl
ddH2O: 37.5 μl
94℃, 5 min. → 94℃, 20 sec. → annealing temp. → 68℃, 1 min. → 68℃, 5 min. → 4℃
36 cycles

4.2 Colony PCR
Taq DNA polymerase (Invitrogen) was used in colony PCR to screen the target
colony. Single colonies were picked and resuspended in 10 μl of Taq polymerase
mixture to perform PCR. The condition of the reaction is as followed:
Taq polymerase mixture:
10× Taq polymerase buffer: 1 μl
10 mM dNTP mixture: 0.2 μl
11
50 mM MgCl
2
: 0.3 μl
Forward primer (10 μM): 0.5 μl
Reverse primer (10 μM): 0.5 μl
Taq DNA polymerase: 0.05 μl
ddH2O: 7.45 μl
94℃, 5 min. → 94℃, 20 sec. → annealing temp. → 72℃, 1 min. → 72℃, 5 min. → 4℃
36 cycles

5. Restriction enzyme digestion
Restriction enzymes XhoⅠand XbaⅠ(Invitrogen) were used for plasmid
construction. The digestion of DNA was carried out under the conditions
recommended by manufacturers.

6. DNA electrophoresis
Appropriate percentage of agarose gel was used to separate DNA fragments. To
make the gel, agarose powder was mixed with 1× TAE buffer to the desired
concentration, and then heated until completely melted. 0.5× TAE buffer was used as
running buffer.
1× TAE buffer (1L): Tris base: 4.84 g
Glacial acetic acid: 1.142 ml
EDTA: 0.372 g
12
7. Recovery of DNA fragments from agarose gel
QIAquick gel extraction kit (QIAGEN) was used to recover DNA fragments
from agarose gel. The excision and recovery of the DNA fragment from agarose gel
was carried out as described in the handbook provided by manufacturer.

8. DNA ligation
T4 DNA ligase (Invitrogen) was used to ligate DNA fragments. Appropriate
amount of linearlized vector and insert DNA fragments were mixed and incubated at
65℃ for 5 min. After the incubation, the mixture was put on ice right away, added
with ligase buffer and ligase, and incubated at 14℃ overnight.

9. Protein expression
9.1 Small scale protein expression
Cells were seeded at appropriate density in 6-well plates the day before
transfection. Before transfection, growth medium was replaced by Opti-MEM. For 1
well, 2 μg of plasmid DNA and 5.5 μl of Lipofectamine
TM
2000 reagent were diluted
in Opti-MEM to 250 μl separately. Subsequently, Lipofectamine
TM
2000- Opti-
MEM mixture was added to DNA- Opti-MEM mixture, mixed gently, and incubated
at room temperature for 20 min to allow the formation of transfection complex. The
transfection complex was added to cells and incubate at 37℃ for 4 hr. After a 4-hour
incubation at 37℃, the medium was replaced by CD 293 medium and the cells were
13
incubated at 37℃ for 4 days. The medium was collected 96 hr. after transfection.
Amicon Ultra (MILLIPORE) was used to concentrate the protein, if necessary.

9.2 Large scale protein expression
Cells were seeded at appropriate density in 225 cm
2
flasks the day before
transfection. Before transfection, growth medium was replaced by FreeStyle 293
medium. For 1 flask, 80 μg of plasmid DNA was diluted in 1.5 ml of 150 mM NaCl,
and 320 μl of PEI (linear polyethylenimine, 25 kD, Polysciences) was also diluted in
150 mM NaCl to 1.5 ml. Diluted PEI was added to diluted DNA solution and the
mixture was incubated at room temperature for 15 min. The transfection complex
was added to cells and incubated at 37℃ for 4~6 hr. After the incubation, the
medium was replaced by FreeStyle 293 medium and incubated at 37℃. The medium
was collected 4 days and 8 days after transfection. Millipore LabScale TFF system
was used to concentration the fusion proteins. Amicon Ultra was used to further
concentrate the proteins, if necessary.

10. SDS-PAGE
10% polyacrylamide gel was used to separate proteins. Protein samples were
mixed with protein loading dye, and boiled for 5~10 min. Samples were loaded into
the wells, and run through the stacking gel at 80 V. After the samples run into
separating gel, the voltage was adjusted to 125 V. The electrophoresis was stopped
when the dye reaches the bottom of the gel.
14
10× SDS-PAGE running buffer: Glycine: 1.92 M
Tris base: 0.25 M
SDS: 1％

11. Western blot
After SDS-PAGE, wet transfer method was used to transfer proteins from gel to
PVDF membrane. The proteins were transferred at 300 mA for 1.5 hr.
10× transfer buffer (1L): Glycine: 144 g
Tris base: 30.28 g
SDS: 1 g

1× transfer buffer (1L): 100 ml of 10× transfer buffer
+ 200 ml of methanol
Add double deionized water to 1 L
After transferring proteins to the membrane, the membrane was blocked by
incubating in 5％ skim milk (skim milk in TBS-T) for 1 hr. The membrane was then
incubated with primary antibody diluted in skim milk at 4℃ overnight. The next day,
the membrane was washed with TBS-T to remove excess antibody. Subsequently,
the membrane was incubated with HRP-conjugated secondary antibody diluted in
skim milk at room temperature for 1 hr. Next, the membrane was washed with TBS-
T and was developed by using ECL
TM
Western Blotting Detection Reagents
(Amersham).
15
TBS-T (1L): Tris: 25 mM
NaCl: 125 mM
Tween-20 : 0.05％

12. In vitro activity assay.
NFS-60 cell line was used to determine the activities of the fusion proteins.
NFS-60 cells were washed with RPMI 1640 medium supplemented with 10% FBS to
remove IL-3. The cells were resuspended and diluted to 8×10
4
cells/ ml. 100 μl of
cell suspension was added to each well of 96-well plate. Fusion protein samples were
diluted in medium (RPMI 1640 medium with 10% FBS), and then 25 μl of diluted
fusion proteins was added to each well. The cells were incubated for 48 hours.
After 48 hours of incubation, CellTiter 96 Aqueous One Solution Cell
Proliferation Assay kit (Promega) was used to measure the proliferation of NFS-60
cells. The reagent contained MTS, a tetrazolium compound, which would be
converted into a formazan product by cells. The formazan product has absorbance
around 490nm. 25 μl of the reagent was added to each well, and the absorbance at
485nm was detected after 1~4 hours of 37℃ incubation.

13. Iodination of Tf.
200 μl of 5 mg/ml Tf was mixed with 50 μl of
125
I and 50 μl of 8 mg/ml
chloramine (dissolved in PBS). The mixture was kept at 0℃ for 5 min, and then 50
μl of 4.8 mg/ml NaMBS (dissolved in PBS) was added. After incubation in ice for
16
another 5 min, 100 μl of 10 mg/ml KI (dissolved in ddH
2
O) was added to stop the
reaction. Sephadex G-50 column was used to separate free iodide from iodinated Tf.

14. TfR binding assay.
Caco-2 cells were seeded in 12-well plates until fully differentiated. Cells were
washed three times with cold PBS and then incubated in DMEM supplemented with
0.1% BSA at 37℃ for 30 min. to remove endogenous Tf. A mixture of
125
I-Tf with
fusion proteins or unlabeled Tf in DMEM supplemented with 0.1% BSA was added
to cells. After incubation at 37℃ for 1 hr, the medium was removed, and the cells
were washed three times with cold PBS. The cells were then lysed with 1 M NaOH,
and the lysates were counted in a gamma counter. BCA assay (Pierce) was
performed to determine the concentration of proteins in each sample to normalize
cell number between each well.

15. Animal study
Male BDF1 mice were used in this study. Proteins were injected subcutaneously
in mice.
Blood samples were collected daily from the tail vein. To get the percentage of
poly-morphonuclear leukocytes, blood samples were treated with ACK lysis buffer
to lyse red blood cells. The samples were then stained with 10 mM Hoechst 33342
and 2.5 μg/ml FITC-conjugated antibody against mouse Gr-1 (Gr-1 is a marker for

17
neutrophil). The percentage of neutrophil (Hoechst
+
, Gr-1
+
) in white blood cells
(Hoechst
+
) was determined by flow cytometry.
To get the total white blood cell count, equal volume of blood samples were
collected, diluted in Isoton®Ⅱ Diluent (Beckman Coulter), and ZAP-OGLOBIN
TM

Ⅱ Lytic Reagent (Beckman Coulter) was added to lyse red blood cells. Coulter cell
counter was used to count the cell number (white blood cells). Absolute neutrophil
count was obtained by multiplying the percentage of neutrophil by total white blood
cell count.
ACK lysis buffer: NH
4
Cl: 0.15 M
KHCO
3
: 10 mM
EDTA: 0.1 mM

18
RESULTS
1. Plasmid construction
1.1 Construction of pcDNA3.1-sTf-G-CSF plasmid.
cDNA of transferrin, and G-CSF were inserted into pcDNA3.1 expression
vector in order to generate eukaryotic expression plasmids for Tf-G-CSF.
Human G-CSF cDNA was obtained by PCR from the plasmid encoding G-CSF-
H4(2)-Tf fusion protein (pcDNA3.0-G-CSF-H4(2)-Tf), and was inserted into the 3’
end of transferrin sequence of pcDNA3.1-Tf, which encoded transferrin with
secretory signal peptide. This new plasmid was named pcDNA3.1-sTf-G-CSF. The
sequences of primers used for producing G-CSF fragment were:
Forward: 5’-GCC TCG AGA CCC CCC TGG GCC CTG C-3’
Reverse: 5’-GCT CTA GAT CAG GGC TGG GCA AGG TG-3’
Annealing temperature for the primers was 58℃.

1.2 Construction of pcDNA3.1-sTf-H4
n
-G-CSF (n=1~3) Plasmid.
To generate Tf-G-CSF fusion protein with H4 linker between the two moieties,
DNA fragment encoding H4 linker (cleaved out by restriction enzyme XhoⅠ from
pcDNA3.0-G-CSF-H4(2)-Tf) was inserted into the XhoⅠ site between transferrin
and G-CSF cDNAs of pcDNA3.1-sTf-G-CSF. Figure 1 is the schematic diagram of
the fusion protein. Colony PCR was used for the preliminary selection of the
plasmids. PCR products from plasmids contain 0, 1, 2, or 3 copies of H4 linker
19
would be 130 bp, 200 bp, 272 bp, and 344 bp, respectively. The sequences of
primers used for colony PCR were:
Forward: 5’-GGC TGT TGG TAA CCT GAG AA-3’
Reverse: 5’-CTC TAA GCA CTT GAG CAG GA-3’
Annealing temperature for the primers was 51℃.
The positive recombinant plasmids screened by colony PCR were sent for
sequencing for further confirmation. Plasmids containing 1, 2, and 3 copies of H4
fragments were isolated, and named pcDNA3.1-sTf-H4
1
-G-CSF, pcDNA3.1-sTf-
H4
2
-G-CSF, and pcDNA3.1-sTf-H4
3
-G-CSF, respectively. Figure 2 is the schematic
drawing of these fusion proteins-expressing plasmids.

20

Figure 1. The schematic diagram of Tf-H4
n
-G-CSF fusion proteins. H4 linker
was inserted between Transferrin and G-CSF moieties. The copy number of the
H4 linker was determined by PCR. PCR products from plasmids contain 0, 1, 2,
or 3 copies of H4 linker were with 130 bp, 200 bp, 272 bp, and 344 bp,
respectively.

Transferrin
C’
H4 Linker
N’
G-CSF
n
21
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-G-CSF
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-H4
1
-G-CSF
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-G-CSF
EcoRV EcoRV
Xho I Xho I
XbaI XbaI
pcDNA3.1-sTf-G-CSF
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-H4
1
-G-CSF
EcoRV EcoRV
Xho I Xho I Xho I
XbaI XbaI
pcDNA3.1-sTf-H4
1
-G-CSF

EcoRV
Xho I
XbaI
pcDNA3.1-sTf-H4
2
-G-CSF pcDNA3.1-sTf-H4
3
-G-CSF
EcoRV
Xho I
XbaI
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-H4
2
-G-CSF
EcoRV EcoRV
Xho I Xho I
XbaI XbaI
pcDNA3.1-sTf-H4
2
-G-CSF pcDNA3.1-sTf-H4
3
-G-CSF
EcoRV
Xho I
XbaI
pcDNA3.1-sTf-H4
3
-G-CSF
EcoRV EcoRV
Xho I Xho I
XbaI XbaI

Figure 2. Schematic drawing of Tf-H4
n
-G-CSF fusion proteins-expressing
plasmids.

CMV promoter
sTf
H4 linker
G-CSF
CMV promoter
sTf
H4 linker
G-CSF
22
2. Expression and identification of sTf-H4
n
-G-CSF (n=0~3) fusion proteins.
pcDNA3.1-sTf-G-CSF, pcDNA3.1-sTf-H4
1
-G-CSF, and pcDNA3.1-sTf-H4
2
-
G-CSF were transfected into HEK-293 cells seeded in 6-well plates. The medium
was collected 4 days after transfection, and then subjected to Western blot assay to
ascertain whether the fusion proteins were produced. Antibodies against human Tf
(1:10,000) and human G-CSF (1:10,000) were used as primary antibodies (2 blots).
Horseradish peroxidase-conjugated anti-goat IgG (1:10,000) and horseradish
peroxidase-conjugated anti-rabbit IgG (1:10,000) antibodies were used as secondary
antibodies. Peroxidase activity was detected by chemiluminescence.
As shown in figure 3 and figure 4, both anti-Tf and anti-G-CSF antibodies
could recognize the fusion proteins. These data confirmed that Tf-H4
n
-G-CSF fusion
proteins were produced.

23

Figure 3. Identification of Tf-H4n-G-CSF fusion proteins by antibody against
human transferrin. 2 μg of each plasmid was transfected into HEK-293 cells
seeded in 6-well plates. Four days after transfection, the conditioned medium
was collected, and subjected to Western blot assay to detect the expression of
the fusion proteins. Lane 1: 50ng of transferrin as a positive control; Lane 2:
50ng of G-CSF; Lane 3: pcDNA3.1 vector; Lane 4: pcDNA3.1-sTf-G-CSF; Lane
5: pcDNA-sTf-H4
1
-G-CSF; Lane 6: pcDNA-sTf-H4
2
-G-CSF.

1 2 3 4 5 6
kD
24

Figure 4. Identification of Tf-H4n-G-CSF fusion proteins by antibody against
human G-CSF. 2 μg of each plasmid was transfected into HEK-293 cells seeded
in 6-well plates. Four days after transfection, the conditioned medium was
collected, and subjected to Western blot assay to detect the expression of the
fusion proteins. Lane 1: 50ng of transferrin; Lane 2: 50ng of G-CSF as a
positive control; Lane 3: pcDNA3.1 vector; Lane 4: pcDNA3.1-sTf-G-CSF;
Lane 5: pcDNA-sTf-H4
1
-G-CSF; Lane 6: pcDNA-sTf-H4
2
-G-CSF.

1 2 3 4 5 6 kD
25
3. H4 linker increases the expression level of Tf-G-CSF fusion proteins
In order to investigate the effect of insertion of H4 linker on fusion proteins
expression, pcDNA3.1-sTf-G-CSF, pcDNA3.1-sTf-H4
1
-G-CSF, pcDNA3.1-sTf-
H4
2
-G-CSF, and pcDNA3.1-sTf-H4
3
-G-CSF were transfected into HEK-293 cells.
The medium was collected 4 days after transfection, and then subjected to Western
blot assay to see whether H4 linker could increase the expression of the fusion
proteins. Anti-transferrin antibody was used to detect the fusion proteins.
As shown in figure 5, the expression level of Tf-G-CSF fusion proteins was
elevated when H4 linker was inserted into the fusion proteins. The protein expression
is dramatically increased when 1 copy of H4 linker is inserted into Tf-G-CSF. The
expression of fusion protein with 2 copies of H4 linker was even higher than that
with 1 copy of linker; however, the difference of expression level between Tf-H4
1
-
G-CSF and Tf-H4
2
-G-CSF was not as much as that between T-G-CSF and Tf-H4
1
-
G-CSF. Moreover, the expression level of Tf-H4
3
-G-CSF was similar to that of Tf-
H4
2
-G-CSF. Therefore, linker with more copies of H4 did not ensure a higher protein
expression.

26

Figure 5. The comparison of the expression level of Tf-H4
n
-G-CSF fusion
proteins (n=0~3). 2 μg of each plasmid was transfected into HEK-293 cells
seeded in 6-well plates. Four days after transfection, the conditioned medium
was collected, and subjected to Western blot assay to detect the expression of
the fusion proteins. Lane 1: 50ng of transferrin; Lane 2: pcDNA3.1; Lane3:
pcDNA3.1-sTf-G-CSF; Lane 4: pcDNA3.1-sTf-H4
1
-G-CSF; Lane 5: pcDNA3.1-
sTf-H4
2
-G-CSF; Lane 6: pcDNA3.1-sTf-H4
3
-G-CSF.

1 2 3 4 5 6 kD
27
4. Tf-H4
n
-G-CSF (n=0~2), and G-CSF-H4
n
-Tf (n=0~3) fusion proteins exhibit
similar in vitro activities.
Plasmids encoding fusion proteins were transfected into HEK-293 cell as
described above. The medium was collected 4 days after transfection, and then
subjected to Western blot assay (antibody against Tf was used as primary antibody)
to detect the proteins. The concentration of the fusion proteins were determined by
using the Quantity One software.
NFS-60 cell line was used to determine the (G-CSF) activities of the fusion
proteins. NFS-60 cells were washed with RPMI 1640 medium supplemented with
10% FBS to remove IL-3. The cells were resuspended and diluted to 8×10
4
cells/ ml.
100 μl of cell suspension was added to each well of 96-well plate. Fusion protein
samples were diluted in medium (RPMI 1640 medium with 10% FBS), and 25 μl of
diluted fusion proteins was added to each well. The final concentrations of the fusion
protein samples in the wells were 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml,
20 ng/ml, 50 ng/ml, and 100 ng/ml G-CSF equivalents. The cells were incubated for
48 hours.
After 48 hours of incubation, CellTiter 96 Aqueous One Solution Cell
Proliferation Assay kit (Promega) was used to measure the proliferation of NFS-60
cells. The reagent contains MTS, a tetrazolium compound, which can be converted
into a formazan product by living cells. The formazan product has absorbance at
around 490 nm. In our studies, 25 μl of the reagent was added to each well, and the
absorbance at 485 nm was detected after 1~4 hours incubation at 37℃.
28
As shown in figure 6, Tf-G-CSF fusion proteins containing 1, 2 and 3 copies of
H4 linker seemed to have similar NFS-60-stimulating activities. The strength of the
maximum response induced by Tf-G-CSF, the fusion protein without H4 linker, was
about 50% of that elicited by the proteins with H4 linkers.
On the other hand, as shown in figure 7, the strength of maximum response
induced by Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf was similar. In other words, the two
fusion proteins exhibited similar in vitro colony-stimulating activities.

29

In vitro Activity of Fusion Proteins (T-G)
-0.5
0
0.5
1
1.5
2
-3 -2 -1 0 1 2 3
Log (G-CSF Equivalent)
Response (A485)
Tf-G-CSF
Tf-H41-G-CSF
Tf-H42-G-CSF
Tf-H43-G-CSF
Positive Control

Figure 6. Evaluation of the in vitro activities of Tf-H4n-G-CSF fusion proteins
(n=0~3). The proliferation of NFS-60 cells was measured via MTS assay.

30

In vitro Activity of Fusion Proteins (T-G, G-T Comparison)
-0.5
0
0.5
1
1.5
2
-3 -2 -1 0 1 2 3
Log (G-CSF Equivalent)
Response (A485)
Tf-G-CSF
Tf-H42-G-CSF
LE (G-CSF-Tf)
G-CSF-H4(2)-Tf
Positive Control

Figure 7. Comparison of the in vitro activities of Tf-G-CSF, Tf-H42-G-CSF, G-
CSF-Tf, and G-CSF-H4(2)-Tf fusion proteins. The proliferation of NFS-60 cells
was measured via MTS assay.

31
5. Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf fusion proteins shows similar binding
abilities to Tf receptor.
To investigate and compare the transferrin receptor binding abilities of sTf-H4
2
-
G-CSF and G-CSF-H4(2)-Tf fusion proteins,
125
I-labeled transferrin was used to
compete with the fusion proteins for binding transferrin receptor.
To generate more fusion proteins, HEK-293 cells were seeded in T-225 cell
culture flasks. Polyethylenimine (PEI) was used for transfection. Medium was
collected 4 and 8 days after incubation. Millipore LabScale TFF system and Amicon
Ultra were used to concentrate the fusion proteins. Western blot (antibody against Tf
as primary antibody) was performed to determine the concentration of the fusion
proteins.
Caco-2 cells were seeded in 12-well plates until fully differentiated.
Endogenous Tf was removed by washing with cold PBS and incubating in DMEM
supplemented with 0.1% BSA at 37℃ for 30 min. Mixtures of 1 μg/ml
125
I-
transferrin with 0.1, 0.3, 1, 3, 10 or 30 μg/ml fusion proteins or unlabeled transferrin
in DMEM supplemented with 0.1% BSA were added to cells. After 37℃ incubation
for 1 hr, the medium was removed, and the cells were then lysed with 1 M NaOH.
The lysates were counted in a gamma counter. BCA assay was performed to
determine the concentration of proteins in each sample to normalize cell number
between each well.
Figure 8 referred to the result of the competition assay. The lower the
radioactive count, the stronger binding abilities toward transferrin receptor the fusion
32
proteins had. The figure showed that the transferrin receptor binding ability of Tf-
H4
2
-G-CSF may be slightly lower than that of G-CSF-H4(2)-Tf; However, opposite
result was also obtained in another independent experiment. The difference in the
binding abilities between the two fusion proteins was quite small in independent
experiments. Therefore, Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf fusion proteins may
possess similar transferrin receptor-binding abilities.

33

In vitro Binding Ability
0
2000
4000
6000
8000
10000
12000
-1.5 -1 -0.5 0 0.5 1 1.5 2
Log (Tf Equivalent)
CPM
Transferrin
Tf-H42-G-CSF
GCSF-H4(2)-Tfxx

Figure 8. Comparison of the TfR binding abilities of Tf-H4
2
-G-CSF and G-CSF-
H4(2)-Tf fusion proteins. To evaluate the transferrin receptor-binding abilities
of the fusion proteins, 1 μg/ml
125
I-labeled transferrin was mixed with 0.1, 0.3, 1,
3, 10 or 30 μg/ml fusion proteins or unlabeled transferrin to compete for the
receptor.

34
6. Tf-H42-G-CSF shows myelopoietic activity in in vivo study.
In order to investigate and compare the in vivo myelopoietic efficacies of Tf-H4
2
-
G-CSF and G-CSF-H4(2)-Tf fusion proteins, proteins were administered to mice.
Absolute neutrophil levels were monitored to estimate the efficacy of the two
proteins. Fusion proteins were produced as described in last section. Male BDF1
mice, which were neutropenia, were used in this study.
5 mg/kg fusion protein or 1 mg/kg G-CSF (Neupogen) were subcutaneously
administered to mice. To determine the effect of the fusion proteins, blood samples
were collected from the tail vein 24, 48, and 72 hr after administration. To get the
percentage of poly-morphonuclear leukocytes, blood samples were treated with ACK
lysis buffer to lyse red blood cells. The samples were then stained with 10 mM
Hoechst 33342 and 2.5 μg/ml FITC-conjugated antibody against mouse Gr-1 (Gr-1 is
a marker for neutrophil). The percentage of neutrophils (Hoechst
+
, Gr-1
+
) in white
blood cells (Hoechst
+
) was determined by flow cytometry. Figure 9.
To get the total white blood cell count, 5 μl of blood samples were collected
from tail veins. The samples were then diluted in 2.5 ml of Isoton®Ⅱ Diluent, and
25 μl of ZAP-OGLOBIN Ⅱ lytic reagent was added to each sample to lyse red blood
cells. Let the lytic reagent to lyse red blood cells for at least 2 min, and then 7.5 ml
of Isoton®Ⅱ Diluent was add to each sample. Coulter cell counter was used to count
the cell number (total white blood cells). Absolute neutrophil count (ANC) was
obtained by multiplying the percentage of neutrophil by total white blood cell count.

35
The result showed that when subcutaneously administered to the mice, both Tf-
H4
2
-G-CSF and G-CSF-H4(2)-Tf fusion proteins showed neutrophil stimulating
activities. Although the two fusion proteins had similar in vitro activities, sTf-H4
2
-G-
CSF exhibited a stronger stimulating effect on neutrophil than G-CSF-H4(2)-Tf.
Figure10 showed that Tf-H4
2
-G-CSF was almost as effective as the positive control,
whereas G-CSF-H4(2)-Tf exhibited lower activity in boosting the level of ANC. On
the other hand, both Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf exhibited prolonged
effects on the neutrophil proliferation in mice.

36

PBS
9.0%
5.0%
(Hoechst)
G-CSF
26.5%
14.2%
(Hoechst)
G-CSF-H4(2)-Tf
14.0%
8.9%
(Hoechst)
Tf-H4
2
-G-CSF
17.0%
7.8%
(Hoechst)
PBS
9.0%
5.0%
(Hoechst)
PBS PBS
9.0%
5.0%
(Hoechst)
G-CSF
26.5%
14.2%
(Hoechst)
G-CSF
26.5%
14.2%
G-CSF G-CSF
26.5%
14.2%
(Hoechst)
G-CSF-H4(2)-Tf
14.0%
8.9%
(Hoechst)
G-CSF-H4(2)-Tf
14.0%
8.9%
G-CSF-H4(2)-Tf G-CSF-H4(2)-Tf
14.0%
8.9%
(Hoechst)
Tf-H4
2
-G-CSF
17.0%
7.8%
(Hoechst)
Tf-H4
2
-G-CSF
17.0%
7.8%
Tf-H4
2
-G-CSF Tf-H4
2
-G-CSF
17.0%
7.8%
(Hoechst)

Figure 9. Evaluation of the percentage of neutrophils in white blood cells is
determined by flow cytometry. Q2 referred to neutrophils (Hoechst
+
, Gr-1
+
),
and Q2 plus Q4 referred to total white blood cells (Hoechst
+
). The percentage of
neutrophils in white blood cells was obtained by dividing Q2 by the sum of Q2
and R4.

37

In vivo (s.c.) Activity of Fusion Proteins
0
0.5
1
1.5
2
2.5
3
3.5
0 2448 7296
Time (hr)
ANC (1,000,000 /ml)
PBS
G-CSF
Tf-H42-G-CSF
G-CSF-H4(2)-Tf

Figure 10. Comparison of the in vivo activities of Tf-H42-G-CSF and G-CSF-
H4(2)-Tf fusion proteins. 5mg/kg of fusion proteins were subcutaneously
administered to BDF1 mice. 1mg/ml of G-CSF was administered as a positive
control. ANC was determined 24, 48 and 72 hr after dosing. N=3 for all groups.

38
DISCUSSION
1. The insertion of helical linker, H4, increases the expression level of Tf-G-
CSF fusion protein.
Results from this study indicate that the insertion of H4 linker is capable to
increase the expression of Tf-G-CSF fusion protein. (Figure 5) There are two
possible ways by which H4 linker could elevate the expression of proteins. First, it is
possible that the fusion of transferrin and G-CSF forms an abnormal conformation in
the region where the two moieties link to each other, thus, the protein may be
thought misfolded and degraded by cells. The insertion of H4 linker pulls the two
moieties apart, and the formation of the motif with abnormal conformation is
minimized. As a result, the expression of fusion proteins is increased. The second
possibility is that H4 linker has some characteristics, such as conformation and
amino acid sequence, which could enhance the expression of fusion proteins.
To test whether H4 linker increases Tf-G-CSF protein expression by avoiding
the formation of the “strange” motif between transferrin and G-CSF, a random linker
should be designed and inserted into Tf-G-CSF. If the random linker could also
boost the fusion protein production, it proves that the increase of protein expression
is due to the removal of misfolded motif.
To investigate whether the increase of Tf-G-CSF protein expression is due to
the intrinsic property of H4 linker, we could change the amino acid residues in H4
linker to generate a linker that is slightly different from H4 linker. If the insertion of

39
this new linker could not elevate the fusion protein expression, it means the elevation
of Tf-G-CSF expression is owing to the special characteristics of H4 linker.

2. The order of transferrin and G-CSF in fusion protein does not have much
influence on the binding of the proteins to transferrin receptor.
In previous study, G-CSF-Tf fusion protein showed lower transferrin receptor
binding ability than native G-CSF. It is thought that the low receptor binding ability
is due to the modification of the N-terminus of transferrin. The N lobe of transferrin
has been reported to strengthen the interaction between transferrin and the target cell.
Therefore, the modification of the transferrin in G-CSF-Tf molecule (G-CSF is
added to the N-terminus of transferrin) may reduce the binding affinity of the fusion
protein to transferrin receptor. It is assumed that Tf-G-CSF, which has no
modification at the N-terminus of transferrin, could exhibit higher receptor binding
ability.
However, in this study, we found that both Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf
exhibit lower transferrin receptor binding abilities than native transferrin, and the
two fusion proteins have similar receptor binding affinities. (Figure 8) This result
implies that the modification at the N-terminus of transferrin may not be responsible
for the low receptor binding abilities of our fusion proteins; there must be other
factors that influence the binding of the fusion proteins to the receptor. One possible
explanation is that the native transferrin we use in the assay is a commercial product,
thus, the process for the preparation of native transferrin is different from that of our
40
fusion proteins. Our fusion proteins may loss part of the receptor binding abilities
during the relatively complicated production process.

3. The in vivo efficacy of the fusion protein does not correlate well with the in
vitro activity.
In this study, although Tf-H4
2
-G-CSF and G-CSF-H4(2)-Tf are found to have
similar in vitro transferrin receptor binding abilities and in vitro G-CSF activities, Tf-
H4
2
-G-CSF exhibits a significantly higher in vivo myelopoietic efficacy than G-CSF-
H4(2)-Tf when administered subcutaneously. In fact, Tf-H4
2
-G-CSF could boost the
ANC as high as positive control does. (Figure 10) It seems that the in vivo activity of
Tf-H4
2
-G-CSF does not correlate with its in vitro activity.
We have no clue why the in vivo myelopoietic efficacies of Tf-H4
2
-G-CSF and
G-CSF-H4(2)-Tf are so different. It is possible that G-CSF-H4(2)-Tf would bind to
other proteins in the body. The binding lowers the level of the “accessible” fusion
protein in the system, and in consequence reduces the effect of the fusion protein.
Another explanation is that Tf-H4
2
-G-CSF may be more resistant to the degradation
machinery in the body than G-CSF-H4(2)-Tf. Therefore, more intact Tf-H4
2
-G-CSF
molecules present in the system and exhibit higher myelopoietic efficacy. More
investigation is required to figure out why the in vivo myelopoietic efficacies of Tf-
H4
2
-G-CSF and G-CSF-H4(2)-Tf are so different.
On the other hand, since Tf-H4
2
-G-CSF has been shown to exhibit comparable
myelopoietic efficacy to G-CSF positive control when subcutaneously administered
41
to BDF1 mice, it is worthy to investigate whether Tf-H4
2
-G-CSF would show
efficacy when administered orally. The work will be carried out in the near future.

42
REFERENCES
12. Aisen P, Leibman A, Pinkowitz RA. The anion-binding functions of
transferrin. Advances in experimental medicine and biology 1974;48(0):125-
40.

22. Bai Y, Ann DK, Shen WC. Recombinant granulocyte colony-stimulating
factor-transferrin fusion protein as an oral myelopoietic agent. Proceedings of
the National Academy of Sciences of the United States of America
2005;102(20):7292-6.

27. Bai Y, Shen WC. Improving the oral efficacy of recombinant granulocyte
colony-stimulating factor and transferrin fusion protein by spacer
optimization. Pharmaceutical research 2006;23(9):2116-21.

11. Bates GW, Schlabach MR. The nonspecific binding of Fe3+ to transferrin in
the absence of synergistic anions. The Journal of biological chemistry
1975;250(6):2177-81.

25. Bedell C. Pegfilgrastim for chemotherapy-induced neutropenia. Clinical
journal of oncology nursing 2003;7(1):55-6, 63-4.

8. Cheng Y, Zak O, Aisen P, Harrison SC, Walz T. Structure of the human
transferrin receptor-transferrin complex. Cell 2004;116(4):565-76.

14. Egyed A. The significance of transferrin-bound bicarbonate in the uptake of
iron by reticulocytes. Biochimica et biophysica acta 1973;304(3):805-13.

24. Eyles JL, Roberts AW, Metcalf D, Wicks IP. Granulocyte colony-stimulating
factor and neutrophils--forgotten mediators of inflammatory disease. Nature
clinical practice 2006;2(9):500-10.

5. Gomme PT, McCann KB, Bertolini J. Transferrin: structure, function and
potential therapeutic actions. Drug discovery today 2005;10(4):267-73.

1. Hamman JH, Enslin GM, Kotze AF. Oral delivery of peptide drugs: barriers
and developments. BioDrugs 2005;19(3):165-77.

16. Hamilton TA, Wada HG, Sussman HH. Identification of transferrin receptors
on the surface of human cultured cells. Proceedings of the National Academy
of Sciences of the United States of America 1979;76(12):6406-10.

43
15. He QY, Mason AB, Nguyen V, MacGillivray RT, Woodworth RC. The
chloride effect is related to anion binding in determining the rate of iron
release from the human transferrin N-lobe. The Biochemical journal
2000;350 Pt 3:909-15.

18. Hemadi M, Kahn PH, Miquel G, El Hage Chahine JM. Transferrin's
mechanism of interaction with receptor 1. Biochemistry 2004;43(6):1736-45.

6. Mietzner TA, Morse SA. The role of iron-binding proteins in the survival of
pathogenic bacteria. Annual review of nutrition 1994;14:471-93.

3. Pakdaman R, Petitjean M, El Hage Chahine JM. Transferrins--a mechanism
for iron uptake by lactoferrin. European journal of biochemistry / FEBS
1998;254(1):144-53.
17. Paterson S, Armstrong NJ, Iacopetta BJ, McArdle HJ, Morgan EH.
Intravesicular pH and iron uptake by immature erythroid cells. Journal of
cellular physiology 1984;120(2):225-32.

23. Roberts AW. G-CSF: a key regulator of neutrophil production, but that's not
all! Growth factors (Chur, Switzerland) 2005;23(1):33-41.

10. Schlabach MR, Bates GW. The synergistic binding of anions and Fe3+ by
transferrin. Implications for the interlocking sites hypothesis. The Journal of
biological chemistry 1975;250(6):2182-8.

13. Schulman HM, Martinez-Medellin J, Sidloi R. The reticulocyte-mediated
release of iron and bicarbonate from transferrin: effect of metabolic inhibitors.
Biochimica et biophysica acta 1974;343(3):529-34.

20. Steverding D. The transferrin receptor of Trypanosoma brucei. Parasitology
international 2000;48(3):191-8.

26. Sung L, Dror Y. Clinical applications of granulocyte-colony stimulating
factor. Front Biosci 2007;12:1988-2002.

2. Vyas SP, Singh A, Sihorkar V. Ligand-receptor-mediated drug delivery: an
emerging paradigm in cellular drug targeting. Critical reviews in therapeutic
drug carrier systems 2001;18(1):1-76.

21. Widera A, Bai Y, Shen WC. The transepithelial transport of a G-CSF-
transferrin conjugate in Caco-2 cells and its myelopoietic effect in BDF1
mice. Pharmaceutical research 2004;21(2):278-84.

44
4. Widera A, Norouziyan F, Shen WC. Mechanisms of TfR-mediated
transcytosis and sorting in epithelial cells and applications toward drug
delivery. Advanced drug delivery reviews 2003;55(11):1439-66.

9. Young SP, Bomford A, Williams R. The effect of the iron saturation of
transferrin on its binding and uptake by rabbit reticulocytes. The Biochemical
journal 1984;219(2):505-10.
7. Zak O, Trinder D, Aisen P. Primary receptor-recognition site of human
transferrin is in the C-terminal lobe. The Journal of biological chemistry
1994;269(10):7110-4.

19. Zaliauskiene L, Kang S, Brouillette CG, Lebowitz J, Arani RB, Collawn JF.
Down-regulation of cell surface receptors is modulated by polar residues
within the transmembrane domain. Molecular biology of the cell
2000;11(8):2643-55.

Abstract (if available)

Abstract In this study, Tf-H42-G-CSF fusion protein was engineered for investigating the effects of H4 helical linker 's on the fusion protein expression and the sequence of transferrin and G-CSF moieties on fusion protein activity. Results from this study indicated that H4 linker not only elevated the expression but also increased the in vitro activity of Tf-G-CSF fusion protein. Tf-H42-G-CSF was also compared with G-CSF-H4(2)-Tf for in vitro TfR binding ability, and in vitro and in vivo activity. The results demonstrated that the change in sequence of transferrin and G-CSF moieties had no effect on the in vitro activity and in vitro TfR binding ability of the fusion protein. However, when administered subcutaneously to BDF1 mice, the fusion protein with transferrin at the N-terminal domain exhibited significantly higher in vivo myelopoietic efficacy than the fusion protein with transferrin at the C-terminal domain.

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Protein aggregation: current scenario and recent developments

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Comparison of the pharmacokinetic profiles between monomeric and oligomeric transferrin

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A novel in-cell lethality-based molecular screening system using a split barnase

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Cationic cell penetrating peptides: characterization of transport properties in epithelial cells and their utilization as delivery systems for protein and peptide drugs

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Computer modeling of protein-peptide interface solvation

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Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH

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Characterization of actin based motility in mammalian cells through LIM and SH3 domain protein 1 (LASP1) and elastin like polypeptide (ELP) fusion protein

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Characterization of the role of LIM and SH3 protein (LASP-1) in endocytosis and actin cytoskeleton remodeling

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Site-specific PEGylation of recombinant immunotoxin αFAP-PE38 for half-life extension

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Proinsulin-transferrin fusion protein as a liver targeting insulin therapeutic in Type 1 diabetes treatment

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Molecular and cellular characterization of alveolar epithelium

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Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

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pH-sensitive cytotoxicity of a cell penetrating peptide fused with a histidine-glutamate co-oligopeptide