# Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT inEscherichia coli

Authors: Jiping Cai; Ruili Wei; Jinwei Cheng
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Vascular endothelial cell growth inhibitor (VEGI) is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC) has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies inE. coli BL21 (DE3), and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.


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## Body

## 1. Introduction

The success of applying an antiangiogenic agent Avastin [1]
in clinical settings for cancer treatment provided the first set of evidence to
support the hypothesis that inhibition of tumor neovascularization can bring
significant benefit to cancer therapy [2]. Because neovascularization under
either physiologic or pathologic conditions is controlled by balance of endogenous
proangiogenic and antiangiogenic factors, an important approach to develop
therapeutic agents for cancers and other angiogenesis-driven diseases is to
use endogenous antiangiogenic factors [3].Vascular endothelial growth inhibitor (VEGI), also known as
tumor necrosis factor superfamily (member 15 (TNFSF15) and TL1A), is a recently
identified antiangiogenic cytokine [4–6]. The VEGI gene is expressed
predominantly in endothelial cells, and the VEGI mRNA is detectable in many
adult human organs, suggesting a physiologic role for this unique gene in the
maintenance of the normal vasculature [7]. Previous reports showed that VEGI is
a potent and specific inhibitor of endothelial cell growth [4–7]. There are
three differential splicing isoforms: VEGI-251, VEGI-192,
and VEGI-174 [8–10]. The initially reported VEGI protein is composed of 174
amino acids [4, 5]. Hydrophobic analysis predicted VEGI-174 to be a type II transmembrane
protein, similar to most tumor necrosis factor (TNF) family members [9]. Recombinant
VEGI comprising only the putative extracellular domain exhibited effective inhibition
of endothelial cell growth but had no effect on the proliferation of breast
tumor cells or smooth muscle cells [4]. Full-length VEGI-174 was found,
however, to have no effect on tumor growth when overexpressed in cancer cells
[5], whereas a secretable fusion protein (sVEGI) comprising a secretion signal peptide
and the putative extracellular domain of VEGI-174 inhibited tumor growth when
overexpressed in cancer cells [5]. This indicates that a solubilized
extracellular domain of VEGI is responsible for its biological activity.Several lines of evidence suggested that tumor growth,
angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP)
activity. The two MMPs most closely correlated with metastasis potential are
the 72 kDa MMP-2 (gelatinase A) and the 92 kDa MMP-9 (gelatinase B) [11, 12]. The
synthetic CTTHWGFTLC-deca peptide (CTT peptide) has been recently described as specific
and selective inhibitor for MMP-2 and -9, which belongs to a novel class of cyclic
tissue-permeable peptides containing an HWGF motif, and could not inhibit other
MMPs or gelatinolytic serine proteinases [13, 14]. CTT peptide has been
evidenced to suppress invasion and migration of both tumor and endothelial
cells in vitro, prevent the growth and invasion of tumors in mice, and
specifically home to tumor vasculature expressing gelatinases (MMP-2 and -9) in
vivo.The objective of our study was to prepare chimeric
protein VEGI (extracellular domain)-CTT
for more potent antitumor therapy. In the present study, we successfully
expressed and purified recombinant protein VEGI-CTT inE. coli. The purified VEGI-CTT showed inhibition of endothelial
cell proliferation and formation of capillary-like tubes by ABAE cells. In
addition, the chimeric protein VEGI-CTT potently reduced the activity of MMP-2 and
MMP-9. The preliminarily in vivo study demonstrated that chimeric protein
VEGI-CTT had more potent antitumor activity than VEGI or CTT peptide against
CA46 human lymphoma xenografts in nude mice.The results described
here suggest that bacterial-expressed VEGI-CTT could permit further study on
its inhibitory activity of tumor growth, invasion or metastasis, molecular
mechanism, and potential application for antiangiogenesis therapy of tumor
clinically.


## 2. Materials and Methods

### 2.1. Materials and Cell Lines

NIH 3T3 cells, lymphoma cell line CA46, and liver cancer
cell line HepG2 were purchased from the American Type Culture Collections
(ATCC, Rockville, Md, USA).
Adult bovine aortic endothelial (ABAE) cells, human umbilical cord vein
endothelial (HUVE) cells, and human vascular smooth muscle (HVSM) cells were
purchased from Clonetics (San Diego,
Calif, USA).
All cells were maintained in their recommended medium.Chelating Sepharose Fast Flow and Superdex75 resin were
obtained from Amersham Pharmacia Biotech (USA), and isopropyl-β-D-thiogalactopyranoside
(IPTG) was purchased from Sigma-Aldrich (USA). The expression vector pET30a(+), which contains a T7 promoter, His-Tag
sequence, and an ampicillin resistance gene, and bacterial strains BL21(DE3)
were purchased from Merck Biosciences (Germany). The plasmid pSW200 which
contains full-length VEGI was a kind gift from Dr. Jingjuan Yao. The PCR reagents
and the molecular biology enzymes were purchased from Takara Bio Inc. (Japan). BCA protein assay reagent
was from Pierce Biotechnology
(Ill, USA). CTTHWGFTLC-deca peptide
was chemically
synthesized. Recombinant VEGI protein was previously generated by our lab and
used as control in the present study.


### 2.2. Construction of Prokaryotic Expression
Vector pET- VEGI-CTT

A prokaryotic expression vector pET-VEGI-CTT was
constructed by subcloning the fragment containing the VEGI-CTT gene. The recombinant
cDNA sequence encoding VEGI-CTT was amplified by PCR from the original plasmid pSW200
containing full-length VEGI cDNA using a forward primer P1: 5′-TCCTCTAGAATGTGTA CAACTCACTGGGGTTTCACACTTTGCGTGAGACAAACTCCCACA-3′ and a reverse primer P2: 5′-CGCCTCGAGCTATAGTAAGAAGGCTCC-3′. The forward primer contained an Xba I site
followed by CTT (CTTHWGFTLC) sequence and the N-terminal sequence of VEGI extracellular
domain. The reverse primer possessed a complementary sequence of the C-terminus
of VEGI extracellular domain, a stop codon, and an Xho I site. The final PCR
product was directly cloned into the pGEM-T vector and then subjected to DNA
sequencing to verify the desired sequence. PCR conditions
consisted of 30 cycles of 94°C for 0.5 minute, 55°C for 0.5 minute,
and 72°C for 1.5 minutes.
The PCR-amplified DNA fragment was separated on 1% agarose gels containing
ethidium bromide, and then subjected to DNA sequencing to verify the desired
sequence. The amplified cDNA fragment was subcloned into E. coli expression vector pET30a(+) to construct the prokaryotic expression vector
pET-VEGI-CTT (Figure 1).Schematic
representation of the cloning steps of VEGI-CTT fragment into the
expression vector pET30a(+). (a) The vector Psw200 containing
full-length VEGI gene was used to amplify recombinant cDNA of human
VEGI-CTT for 483 bp by PCR. The recombinant cDNA of human VEGI-CTT
was cloned into pGEM-T, and produced pGEM-T-VEGI-CTT. (b) pET30a(+)
vector with His-tag upstream of the multiple cloning site that
contains XbaI and XhoI.
(c) pET30a(+) carrying XbaI-XhoI fragment of VEGI-CTT
to produce pET30a(+)-VEGI-CTT. The resulting fusion gene contains
internal His-tag and VEGI-CTT.
(a)(b)(c)


### 2.3. Expression and Purification of Chimeric Protein

E. coli BL21(DE3) was transformed with expression plasmid pET-VEGI-CTT.
Bacterial cultures were incubated at 37°C in LB growth medium with antibiotic selection
(100 ng/mL ampicillin), and grown until early log phase (A600 = 0.4–0.8). The
targeted protein expression was induced at 37°C by the addition of IPTG to a final concentration
of 1μM, and incubated with shaking for 2–8 hours. The bacteria were collected
by centrifugation at 4000 rpm for 30 minutes.The pellet was resuspended in 20μM Tris-HCl (pH 8), sonicated at maximal output, and
centrifuged at 12000 rpm for 30 minutes at 4°C. The pellet was
an inclusion body which was denatured by 8 M urea for 2 hours at room
temperature, then centrifuged at 10000 rpm for 30 minutes at 4°C, and the supernatant was filtered (0.45μM pores) and loaded at room temperature onto an His-Ni2+ metal affinity column pre-equilibrated with 8 M urea, 100μM NaH2PO4, 10μM Tris, and 10μM imidazole. Refolding of the bound protein is performed
by the use of a linear 8–0 M urea gradient,
starting with the equilibrating buffer above and finishing with 50 column volumes of equilibrating buffer without urea, then eluted with 5 column volumes of eluting buffer (20μM phosphate, 500μM NaCl, and 300μM imidazole). Protein
concentration was determined by bicinchoninic acid (BCA) assay.


### 2.4. SDS-PAGE and Western blotting

To confirm and compare the quality of recombinant protein
expression, 10–12μL from each fraction were analyzed by SDS-PAGE (12% gels)
followed by staining with Coomassie brilliant blue R-250. For western blotting analysis,
proteins were electrotransferred to PVDF (Amersham-Pharmacia Biotech) membranes
using Mini Trans-Blot cell (Bio-Rad) following manufacturer’s instruction, blocked
in 0.5% BSA in TBS (100μM Tris, 0.9% NaCl, pH 7.5), and immunoreacted with a 1 : 5000 dilution of mouse anti-His
monoclonal antibody (TIANGEN, Beijing, China), followed by an HRP-labeled goat antimouse IgG
(Zymed, USA). Chromogenic-based detection was performed using DAB as a staining
substrate.


### 2.5. Cell Proliferation Assay

Cells were seeded in triplicate at 3000–5000/well in 96-well
plates in IMEM (GIBCO, Gaithersburg,
Md, USA) containing 10% fetal calf serum
(FCS), and cultured at 37°C with
5% CO2. ABAE and HUVE cell culture media contained additional 1 ng/mL and 6
ng/mL FGF-2, respectively. VEGI-CTT ， VEGI, or CTT peptide
in different concentrations
was added to each well, and the blank control wells were only added with equal
volume of PBS. The media were changed once on day 3. The number of viable cells
was determined on day 6 by using a Coulter (Hialeah, Fla, USA) counter.


### 2.6. In Vitro Angiogenesis Assay

Quantitative assessment of capillary-like tube formation
by endothelial cells cultured on collagen gels was carried out as described previously
[4]. To prepare a three-dimensional collagen gel, 0.5 mL chilled IMEM with 10% FBS and 0.7 mg/mL rat tail type I collagen (BD, Franklin Lakes, NJ, USA) was added to each well of a 24-well plate. After formation of the collagen gel (about 1-2 mm thickness), ABAE cells were seeded at 5×104 cells/well.
The cultures were maintained in IMEM with 10% FCS, 1 ng/mL of FGF-2, 5% CO2, and 37°C for 72 hours. The media were replaced
with fresh media omitting FGF-2, and were cultured for 48 hours. Then the media
were replaced with fresh media containing 20 ng/mL of FGF-2. The cultures were
maintained at 37°C for 48 hours.
The gels were then fixed with cold methanol. The abundance of the
capillary-like structures formed by ABAE cells was determined by
computer-assisted image analysis. The relative intensity of the capillary-like
structures was measured as the ratio of the total length of the tubular
structures over the total areas measured (mm/mm2).


### 2.7. In Vitroβ-casein Degradation Assay

Inhibition of MMP-9 and MMP-2 by the chimeric protein
VEGI-CTT was measured using casein degradation assay (13). Subsequently, MMP-2
(2.5 ug) or MMP-9 (2.5 ug) was run on a 10% SDS-PAGE containing 2 mg/mLβ-casein. The gel was first washed in Triton X-100
containing buffer to remove SDS, and it was cut into slices that were immersed
into the solutions containing different concentrations of VEGI-CTT, VEGI, or CTT peptide. After
incubation for 48 hours at 37°C,
the gels were stained with Coomassie Blue and scanned, and the digested areas were
quantitated using image analysis (Global Lab Image 3.2; Data Translation Inc.
and Acuity Imaging Inc., Marlboro, Mass, USA).


### 2.8. In Vivo Study

Six- to eight-week-old female nude mice were obtained from
the Planned Parenthood Research Institute, Shanghai, China.
All animals in this study were housed under pathogen-free conditions and were
maintained in accordance with guidelines of the Committee on Animals of the Second Military Medical University,
Shanghai, China.The xenograft CA46 model was carried out as described [15].
In brief, xenografts were allowed to be established to an average size of 50–100 mm3,
after which mice were randomized into various conditional groups. VEGI-CTT
protein, VEGI protein, or CTT peptide was given to each mouse at designated dose
via intraperitoneal injection at a frequency of twice a week. Each mouse was
measured for tumor size using a caliper on alternate days. Animal body weight
and any sign of morbidity were also closely monitored. The treatment lasted for
2 weeks; at predesigned points mice were killed, and tumor xenografts were
extirpated, weighed, and correlated with the tumor size measurement.


### 2.9. Statistical Analysis

Between-group differences were determined using a
Student’st-test, and multiple treatment groups were compared within individual
experiments by analysis of variance or the Kruskal-Wallis test. P values less than .05 were considered to
be statistically significant.


## 2.1. Materials and Cell Lines

NIH 3T3 cells, lymphoma cell line CA46, and liver cancer
cell line HepG2 were purchased from the American Type Culture Collections
(ATCC, Rockville, Md, USA).
Adult bovine aortic endothelial (ABAE) cells, human umbilical cord vein
endothelial (HUVE) cells, and human vascular smooth muscle (HVSM) cells were
purchased from Clonetics (San Diego,
Calif, USA).
All cells were maintained in their recommended medium.Chelating Sepharose Fast Flow and Superdex75 resin were
obtained from Amersham Pharmacia Biotech (USA), and isopropyl-β-D-thiogalactopyranoside
(IPTG) was purchased from Sigma-Aldrich (USA). The expression vector pET30a(+), which contains a T7 promoter, His-Tag
sequence, and an ampicillin resistance gene, and bacterial strains BL21(DE3)
were purchased from Merck Biosciences (Germany). The plasmid pSW200 which
contains full-length VEGI was a kind gift from Dr. Jingjuan Yao. The PCR reagents
and the molecular biology enzymes were purchased from Takara Bio Inc. (Japan). BCA protein assay reagent
was from Pierce Biotechnology
(Ill, USA). CTTHWGFTLC-deca peptide
was chemically
synthesized. Recombinant VEGI protein was previously generated by our lab and
used as control in the present study.


## 2.2. Construction of Prokaryotic Expression
Vector pET- VEGI-CTT

A prokaryotic expression vector pET-VEGI-CTT was
constructed by subcloning the fragment containing the VEGI-CTT gene. The recombinant
cDNA sequence encoding VEGI-CTT was amplified by PCR from the original plasmid pSW200
containing full-length VEGI cDNA using a forward primer P1: 5′-TCCTCTAGAATGTGTA CAACTCACTGGGGTTTCACACTTTGCGTGAGACAAACTCCCACA-3′ and a reverse primer P2: 5′-CGCCTCGAGCTATAGTAAGAAGGCTCC-3′. The forward primer contained an Xba I site
followed by CTT (CTTHWGFTLC) sequence and the N-terminal sequence of VEGI extracellular
domain. The reverse primer possessed a complementary sequence of the C-terminus
of VEGI extracellular domain, a stop codon, and an Xho I site. The final PCR
product was directly cloned into the pGEM-T vector and then subjected to DNA
sequencing to verify the desired sequence. PCR conditions
consisted of 30 cycles of 94°C for 0.5 minute, 55°C for 0.5 minute,
and 72°C for 1.5 minutes.
The PCR-amplified DNA fragment was separated on 1% agarose gels containing
ethidium bromide, and then subjected to DNA sequencing to verify the desired
sequence. The amplified cDNA fragment was subcloned into E. coli expression vector pET30a(+) to construct the prokaryotic expression vector
pET-VEGI-CTT (Figure 1).Schematic
representation of the cloning steps of VEGI-CTT fragment into the
expression vector pET30a(+). (a) The vector Psw200 containing
full-length VEGI gene was used to amplify recombinant cDNA of human
VEGI-CTT for 483 bp by PCR. The recombinant cDNA of human VEGI-CTT
was cloned into pGEM-T, and produced pGEM-T-VEGI-CTT. (b) pET30a(+)
vector with His-tag upstream of the multiple cloning site that
contains XbaI and XhoI.
(c) pET30a(+) carrying XbaI-XhoI fragment of VEGI-CTT
to produce pET30a(+)-VEGI-CTT. The resulting fusion gene contains
internal His-tag and VEGI-CTT.
(a)(b)(c)


## 2.3. Expression and Purification of Chimeric Protein

E. coli BL21(DE3) was transformed with expression plasmid pET-VEGI-CTT.
Bacterial cultures were incubated at 37°C in LB growth medium with antibiotic selection
(100 ng/mL ampicillin), and grown until early log phase (A600 = 0.4–0.8). The
targeted protein expression was induced at 37°C by the addition of IPTG to a final concentration
of 1μM, and incubated with shaking for 2–8 hours. The bacteria were collected
by centrifugation at 4000 rpm for 30 minutes.The pellet was resuspended in 20μM Tris-HCl (pH 8), sonicated at maximal output, and
centrifuged at 12000 rpm for 30 minutes at 4°C. The pellet was
an inclusion body which was denatured by 8 M urea for 2 hours at room
temperature, then centrifuged at 10000 rpm for 30 minutes at 4°C, and the supernatant was filtered (0.45μM pores) and loaded at room temperature onto an His-Ni2+ metal affinity column pre-equilibrated with 8 M urea, 100μM NaH2PO4, 10μM Tris, and 10μM imidazole. Refolding of the bound protein is performed
by the use of a linear 8–0 M urea gradient,
starting with the equilibrating buffer above and finishing with 50 column volumes of equilibrating buffer without urea, then eluted with 5 column volumes of eluting buffer (20μM phosphate, 500μM NaCl, and 300μM imidazole). Protein
concentration was determined by bicinchoninic acid (BCA) assay.


## 2.4. SDS-PAGE and Western blotting

To confirm and compare the quality of recombinant protein
expression, 10–12μL from each fraction were analyzed by SDS-PAGE (12% gels)
followed by staining with Coomassie brilliant blue R-250. For western blotting analysis,
proteins were electrotransferred to PVDF (Amersham-Pharmacia Biotech) membranes
using Mini Trans-Blot cell (Bio-Rad) following manufacturer’s instruction, blocked
in 0.5% BSA in TBS (100μM Tris, 0.9% NaCl, pH 7.5), and immunoreacted with a 1 : 5000 dilution of mouse anti-His
monoclonal antibody (TIANGEN, Beijing, China), followed by an HRP-labeled goat antimouse IgG
(Zymed, USA). Chromogenic-based detection was performed using DAB as a staining
substrate.


## 2.5. Cell Proliferation Assay

Cells were seeded in triplicate at 3000–5000/well in 96-well
plates in IMEM (GIBCO, Gaithersburg,
Md, USA) containing 10% fetal calf serum
(FCS), and cultured at 37°C with
5% CO2. ABAE and HUVE cell culture media contained additional 1 ng/mL and 6
ng/mL FGF-2, respectively. VEGI-CTT ， VEGI, or CTT peptide
in different concentrations
was added to each well, and the blank control wells were only added with equal
volume of PBS. The media were changed once on day 3. The number of viable cells
was determined on day 6 by using a Coulter (Hialeah, Fla, USA) counter.


## 2.6. In Vitro Angiogenesis Assay

Quantitative assessment of capillary-like tube formation
by endothelial cells cultured on collagen gels was carried out as described previously
[4]. To prepare a three-dimensional collagen gel, 0.5 mL chilled IMEM with 10% FBS and 0.7 mg/mL rat tail type I collagen (BD, Franklin Lakes, NJ, USA) was added to each well of a 24-well plate. After formation of the collagen gel (about 1-2 mm thickness), ABAE cells were seeded at 5×104 cells/well.
The cultures were maintained in IMEM with 10% FCS, 1 ng/mL of FGF-2, 5% CO2, and 37°C for 72 hours. The media were replaced
with fresh media omitting FGF-2, and were cultured for 48 hours. Then the media
were replaced with fresh media containing 20 ng/mL of FGF-2. The cultures were
maintained at 37°C for 48 hours.
The gels were then fixed with cold methanol. The abundance of the
capillary-like structures formed by ABAE cells was determined by
computer-assisted image analysis. The relative intensity of the capillary-like
structures was measured as the ratio of the total length of the tubular
structures over the total areas measured (mm/mm2).


## 2.7. In Vitroβ-casein Degradation Assay

Inhibition of MMP-9 and MMP-2 by the chimeric protein
VEGI-CTT was measured using casein degradation assay (13). Subsequently, MMP-2
(2.5 ug) or MMP-9 (2.5 ug) was run on a 10% SDS-PAGE containing 2 mg/mLβ-casein. The gel was first washed in Triton X-100
containing buffer to remove SDS, and it was cut into slices that were immersed
into the solutions containing different concentrations of VEGI-CTT, VEGI, or CTT peptide. After
incubation for 48 hours at 37°C,
the gels were stained with Coomassie Blue and scanned, and the digested areas were
quantitated using image analysis (Global Lab Image 3.2; Data Translation Inc.
and Acuity Imaging Inc., Marlboro, Mass, USA).


## 2.8. In Vivo Study

Six- to eight-week-old female nude mice were obtained from
the Planned Parenthood Research Institute, Shanghai, China.
All animals in this study were housed under pathogen-free conditions and were
maintained in accordance with guidelines of the Committee on Animals of the Second Military Medical University,
Shanghai, China.The xenograft CA46 model was carried out as described [15].
In brief, xenografts were allowed to be established to an average size of 50–100 mm3,
after which mice were randomized into various conditional groups. VEGI-CTT
protein, VEGI protein, or CTT peptide was given to each mouse at designated dose
via intraperitoneal injection at a frequency of twice a week. Each mouse was
measured for tumor size using a caliper on alternate days. Animal body weight
and any sign of morbidity were also closely monitored. The treatment lasted for
2 weeks; at predesigned points mice were killed, and tumor xenografts were
extirpated, weighed, and correlated with the tumor size measurement.


## 2.9. Statistical Analysis

Between-group differences were determined using a
Student’st-test, and multiple treatment groups were compared within individual
experiments by analysis of variance or the Kruskal-Wallis test. P values less than .05 were considered to
be statistically significant.


## 3. Results

### 3.1. Construction of Prokaryotic Expression
Vector pET- VEGI-CTT

The vector pSW200 containing full-length VEGI gene was used
to amplify recombinant cDNA of human VEGI-CTT by PCR. Analysis of 1% agarose
gels revealed that the resulting fragment was≈490 bp (Figure 2(a)), and then VEGI-CTT cDNA was cloned
into pGEM-T vector for sequencing, which showed a fragment with 486 bp in
length.Construction of prokaryotic expression vector pET-VEGI-CTT. (a)
DNA fragments of VEGI-CTT were amplified by PCR. Lane 1: 100 bp
markers. Lane 2: VEGI-CTT fragment of 486 bp. (b) Restriction enzyme
analysis of the prokaryotic expression vector pET-VEGI-CTT. Lane 1: 100 bp markers; lane 2: pET-VEGI-CTT digested by Xba I and Xho
I.
(a)(b)The amplified cDNA fragment was subcloned into pET30a(+) to produce a prokaryotic expression
vector pET-VEGI-CTT; subsequently the resulting pET-VEGI-CTT was identified by
digestion of restriction enzymes XbaI and XhoI, which revealed that the cDNA of
VEGI-CTT was correctly inserted into the vector pET30a(+) (Figure2(b)).


### 3.2. Expression, Refolding, and Purification of Chimeric Protein VEGI-CTT

The resulting plasmids pET30a(+)-VEGI-CTT and the controls were transformed into
theE. coli BL21(DE3). This
construction of pET30a(+)-VEGI-CTT
should produce a recombinant protein of approximately 22 kDa. Induction of the
strong T7 promoter controlling the expression of VEGI-CTT in BL21(DE3) with 1μM IPTG produced recombinant VEGI-CTT after
2 hours. It seemed that there was no difference in expression levels from 2 to
6 hours at 37°C (Figure 3(a)). The recombinant protein is the major band present at all time points of
induction.Expression analysis of recombinant VEGI-CTT in BL21(DE3). (a)
Time course analysis of recombinant VEGI-CTT induction in BL21(DE3)
induced by 1μM IPTG. Protein samples were prepared from the
inclusion-body fraction of induced bacterial cells at time points of
2 hours (lane 3), 4 hours (lane 4), 6 hours (lane 5), and 8 hours
(lane 6) of the
induction, and were resolved on 12% SDS-PAGE. Lane 1: low molecular weight
protein markers; lane 2: uninduced control. No difference was found
from lane 3 to lane 6 at 2–8 hours. (b) SDS-PAGE analysis of
expression and location of the fusion protein VEGI-CTT in the
cellular fractions of E. coli. Lane 1: eluted
fraction under renaturing conditions with 500μM imidazole; lane 2:
low molecular weight protein markers; lane 3: the inclusion-body
fraction; lane 4: supernatant of cell lysates.
(a)(b)To determine the
location of the recombinant VEGI-CTT in the cellular fractions ofE. coli, the cytosolic, inclusion-body,
periplasmic, and media fractions were tested. The recombinant protein was found
only in the inclusion-body fraction; almost no VEGI-CTT protein was observed in
the soluble fraction (Figure 3(b)). Likewise, no indication of secreted or
transferred protein was observed in the media or periplasmic fractions. No effect
on the solubility of the recombinant protein was observed by decreasing the
incubation temperature or by slowing the expression by decreasing the
concentration of the IPTG (results are not shown).Following the successful overproduction of recombinant
VEGI-CTT in the inclusion-body fraction ofE.
coli, we attempted to use the presence of the internal His-tag in the
fusion protein for renaturation and purification on immobilized metal ion-affinity
chromatography (Ni2+-chelating) column. The recombinant VEGI-CTT in
inclusion body was denatured by 8 M urea. A step-down urea concentration strategy (a linear gradient from 8 to 0 M urea) was applied to the VEGI-CTT
on-column refolding process. The VEGI-CTT fusion proteins were eluted with 300μM imidazole, with purity being greater
than 90%, and they were of the expected 22 kDa (Figure 3(b)). By this strategy,
a stable final amount of the obtained purified recombinant VEGI-CTT was
approximately 25.5 mg/L of initial bacterial broth. The refolding and
purification yield of recombinant VEGI-CTT was about 16.5% (Table 1).Table 1
Purification of chimeric protein VEGI-CTT from inclusion
bodies (from 1 L ofE. coli culture and 4.6 g wet weight cells).
StepsTotal  protein (mg)Step yield (%)Overall yield (%)Cell
lysate304——Inclusion
body155100100Solubilization
(denaturation buffer containing 8 M urea)96.362.162.1Refolding
and
affinity
purification25.526.616.5To insure that the recombinant protein we had expressed
and renatured was the protein we designed, the VEGI-CTT was subjected to western
blotting analysis using His-tag monoclonal antibody. As shown in Figure4, the
purified recombinant protein is His-immunoreactive and, as expected, it has a
molecular weight of approximately 22 kDa.Figure 4
Western blotting analysis of the renatured, purified recombinant
protein VEGI-CTT. Ten micrograms of VEGI-CTT were
applied into 12% SDS-PAGE, and then electrotransferred to a PVDF membrane.
Western blotting analysis was performed using mouse anti-His-Tag
monoclonal antibody (Lane 2). The membrane was incubated with the
second antibody: HRP-labeled goat antimouse IgG. Lane 1: prestained
molecular weight protein markers. Lane 2: fusion protein VEGI-CTT of
22 kDa.


### 3.3. Chimeric Protein VEGI-CTT Inhibits the
Proliferation of the Endothelial Cells in Vitro

The bioactivity of VEGI-CTT was determined
by the proliferation of ABAE and HUVE cells in vitro. As shown in Figure5(a), the
protein was found to preferentially inhibit the FGF-2-induced proliferation of
ABAE and HUVE cells in a dose-dependent manner, whereas it could not inhibit
the growth of HVSM, liver cancer HepG2 or NIH 3T3 cells. The half-maximum
inhibitory concentrations (IC50) of VEGI-CTT
for ABAE and HUVE cells were about 25 ng/mL and 100 ng/mL, respectively. The inhibition
effect of VEGI-CTT on endothelial cells was similar to that of VEGI alone or of
VEGI and CTT combined treatment at a concentration of 40 ng/mL. No effect on the
proliferation of the endothelial cells or cancer cells was observed in the
group of CTT treatment (Figure 5(b)). These
results suggest that recombinant protein VEGI-CTT, which is bioactive, could specifically
inhibit proliferation of endothelial cells when compared with other tested cell
types.Specific inhibition of endothelial cell proliferation by chimeric
protein VEGI-CTT. Cells were seeded in triplicate at
3000–5000 cells/well in 96-well plates. Culture conditions
are described in Section2. The media were changed once on day 3.
The number of viable cells was determined on day 6 by using a
Coulter counter. Mean values are presented as a function of VEGI-CTT
or other agents’ concentrations.  P*<.05 versus
that of PBS group.
(a)(b)


### 3.4. Inhibition of in Vitro Angiogenesis by
Chimeric Protein VEGI-CTT

The antiangiogenic activity of the recombinant VEGI-CTT
was examined with an in vitro angiogenesis model. In this model, when
recombinant VEGI-CTT was added to the cell cultures together with FGF-2,
inhibition of the formation of capillary-like tubes by ABAE cells was observed (Figure6). The relative intensities of the tubules were
determined by using computer-assisted image analysis. The IC50 value
for the inhibition was found to be approximately 40 ng/mL. VEGI used alone or VEGI and CTT combined treatment also could inhibit the formation
of capillary-like tubes by ABAE cells similar to those of VEGI-CTT, whereas
the antiangiogenic activity of CTT was not observed (Figure 6). These results further
demonstrated that recombinant protein VEGI-CTT had a potent activity of antiangiogenesis
through inhibiting the proliferation of endothelial cells.Figure 6
Inhibition of formation of capillary-like tubules by adult bovine
aortic endothelial (ABAE) cells in collagen gels. The experiments
were carried out in triplicate. At least 9 image areas were analyzed
for each VEGI concentration. Mean values and standard deviations are
presented as a function of VEGI concentration.P*<.05 versus that of PBS group.


### 3.5. VEGI-CTT Potently Reduced the Activity of MMP-2 and MMP-9

CTT is a recently described cyclic peptide collagenase inhibitor of MMP-2 and MMP-9. To determine
whether the recombinant protein we had expressed and renatured also could efficiently
inhibit the activity of MMP-2 and MMP-9, metalloproteinase inhibitory activity
of VEGI-CTT was assessed by casein degradation assay. In order to precisely
compare the bioactivity between recombinant protein VEGI-CTT and CTT peptide, molar
concentration was used here. As shown in Figure7, degradation of β-casein by MMP-9 or MMP-2 was inhibited by VEGI-CTT, similar
to that of CTT or both CTT and VEGI. Moreover, the inhibitory effect of
VEGI-CTT occurred in a dose-dependent
manner. For MMP-2, the fusion protein VEGI-CTT
inhibited MMP-2 with an IC50 of 150 ng/mL (equal to 7.5μM). Caseinolysis by MMP-9 was similarly inhibited by the protein
at high concentrations with an IC50 of 650 ng/mL (equal to 30μM). The differences of IC50 for MMP-2 and MMP-9
may be due to the different avidity of VEGI-CTT with them. However,
VEGI alone could not inhibit the degradation
of β-casein by MMP-9 or MMP-2. The data indicated that recombinant
protein VEGI-CTT had similar activity to the CTT peptide as previously reported,
and could potently
reduce the activity of MMP-2 and MMP-9 in a dose-dependent manner.Inhibition of caseinolytic activity by the chimeric protein
VEGI-CTT. MMP-2  and MMP-9 enzymes were incubated with 2 mg/mLβ-casein in
the absence or presence of VEGI-CTT. Proteins were separated on a 10% SDS-polyacrylamide
gel, and detected with Serve blue R. For details, see Section 2.
Inhibition of MMP-2 and MMP-9 mediated β-casein degradation by
VEGI-CTT at different concentrations with apparent IC50 of 7.5μM
and 30μM, respectively.
(a)(b)


### 3.6. Xenograft Antitumor Activity

We extended our analysis of the antitumor activity of chimeric
protein VEGI-CTT using mouse xenograft models. Cultured CA46 lymphoma cells
were implanted subcutaneously into nude mice, the agent treatment started upon tumor
xenograft development, and the tumor growth was monitored for 2 weeks.
Subcutaneous inoculation of1×107 CA46 cells resulted in
aggressively growing tumors in PBS treatment group (Figure 8). Tumor-bearing mice
were treated with predetermined doses of chimeric protein VEGI-CTT, and
significant reduction in tumor burden was observed. Treatment with 50 ug/mouse VEGI-CTT
twice a week reduced tumor growth by 82.2% compared with animals in the PBS-treated
group. In contrast, treatment with 50 ug/mouse VEGI or CTT peptide alone merely
resulted in 49.2% and 35.1% reduction, respectively. In addition, compared with
VEGI-CTT treatment, combined treatment with 50 ug/mouse VEGI and CTT peptide had
a relatively low antitumor activity but also significantly reduced tumor growth
by 62.9%. These results establish chimeric protein VEGI-CTT as an effective
inhibitor of lymphoma tumor growth in vivo, and thus it had a more potent
antitumor activity than VEGI, CTT, or the combination of both.Figure 8
Effect of chimeric protein VEGI-CTT in treating CA46 human
lymphoma xenograft-bearing nude mice. Nude mice were inoculated with
CA46 human lymphoma cells, and the treatment started upon tumor
xenograft establishment in nude mice. Tumor xenograft-bearing nude
mice were injected with indicated doses of VEGI-CTT, VEGI, CTT, or
VEGI and CTT combined treatment for two weeks, and tumor volumes
were measured on alternate days. Mean values and standard deviations
are presented as tumor volumes in each group (n=9). The experiments
were repeated with similar results.


## 3.1. Construction of Prokaryotic Expression
Vector pET- VEGI-CTT

The vector pSW200 containing full-length VEGI gene was used
to amplify recombinant cDNA of human VEGI-CTT by PCR. Analysis of 1% agarose
gels revealed that the resulting fragment was≈490 bp (Figure 2(a)), and then VEGI-CTT cDNA was cloned
into pGEM-T vector for sequencing, which showed a fragment with 486 bp in
length.Construction of prokaryotic expression vector pET-VEGI-CTT. (a)
DNA fragments of VEGI-CTT were amplified by PCR. Lane 1: 100 bp
markers. Lane 2: VEGI-CTT fragment of 486 bp. (b) Restriction enzyme
analysis of the prokaryotic expression vector pET-VEGI-CTT. Lane 1: 100 bp markers; lane 2: pET-VEGI-CTT digested by Xba I and Xho
I.
(a)(b)The amplified cDNA fragment was subcloned into pET30a(+) to produce a prokaryotic expression
vector pET-VEGI-CTT; subsequently the resulting pET-VEGI-CTT was identified by
digestion of restriction enzymes XbaI and XhoI, which revealed that the cDNA of
VEGI-CTT was correctly inserted into the vector pET30a(+) (Figure2(b)).


## 3.2. Expression, Refolding, and Purification of Chimeric Protein VEGI-CTT

The resulting plasmids pET30a(+)-VEGI-CTT and the controls were transformed into
theE. coli BL21(DE3). This
construction of pET30a(+)-VEGI-CTT
should produce a recombinant protein of approximately 22 kDa. Induction of the
strong T7 promoter controlling the expression of VEGI-CTT in BL21(DE3) with 1μM IPTG produced recombinant VEGI-CTT after
2 hours. It seemed that there was no difference in expression levels from 2 to
6 hours at 37°C (Figure 3(a)). The recombinant protein is the major band present at all time points of
induction.Expression analysis of recombinant VEGI-CTT in BL21(DE3). (a)
Time course analysis of recombinant VEGI-CTT induction in BL21(DE3)
induced by 1μM IPTG. Protein samples were prepared from the
inclusion-body fraction of induced bacterial cells at time points of
2 hours (lane 3), 4 hours (lane 4), 6 hours (lane 5), and 8 hours
(lane 6) of the
induction, and were resolved on 12% SDS-PAGE. Lane 1: low molecular weight
protein markers; lane 2: uninduced control. No difference was found
from lane 3 to lane 6 at 2–8 hours. (b) SDS-PAGE analysis of
expression and location of the fusion protein VEGI-CTT in the
cellular fractions of E. coli. Lane 1: eluted
fraction under renaturing conditions with 500μM imidazole; lane 2:
low molecular weight protein markers; lane 3: the inclusion-body
fraction; lane 4: supernatant of cell lysates.
(a)(b)To determine the
location of the recombinant VEGI-CTT in the cellular fractions ofE. coli, the cytosolic, inclusion-body,
periplasmic, and media fractions were tested. The recombinant protein was found
only in the inclusion-body fraction; almost no VEGI-CTT protein was observed in
the soluble fraction (Figure 3(b)). Likewise, no indication of secreted or
transferred protein was observed in the media or periplasmic fractions. No effect
on the solubility of the recombinant protein was observed by decreasing the
incubation temperature or by slowing the expression by decreasing the
concentration of the IPTG (results are not shown).Following the successful overproduction of recombinant
VEGI-CTT in the inclusion-body fraction ofE.
coli, we attempted to use the presence of the internal His-tag in the
fusion protein for renaturation and purification on immobilized metal ion-affinity
chromatography (Ni2+-chelating) column. The recombinant VEGI-CTT in
inclusion body was denatured by 8 M urea. A step-down urea concentration strategy (a linear gradient from 8 to 0 M urea) was applied to the VEGI-CTT
on-column refolding process. The VEGI-CTT fusion proteins were eluted with 300μM imidazole, with purity being greater
than 90%, and they were of the expected 22 kDa (Figure 3(b)). By this strategy,
a stable final amount of the obtained purified recombinant VEGI-CTT was
approximately 25.5 mg/L of initial bacterial broth. The refolding and
purification yield of recombinant VEGI-CTT was about 16.5% (Table 1).Table 1
Purification of chimeric protein VEGI-CTT from inclusion
bodies (from 1 L ofE. coli culture and 4.6 g wet weight cells).
StepsTotal  protein (mg)Step yield (%)Overall yield (%)Cell
lysate304——Inclusion
body155100100Solubilization
(denaturation buffer containing 8 M urea)96.362.162.1Refolding
and
affinity
purification25.526.616.5To insure that the recombinant protein we had expressed
and renatured was the protein we designed, the VEGI-CTT was subjected to western
blotting analysis using His-tag monoclonal antibody. As shown in Figure4, the
purified recombinant protein is His-immunoreactive and, as expected, it has a
molecular weight of approximately 22 kDa.Figure 4
Western blotting analysis of the renatured, purified recombinant
protein VEGI-CTT. Ten micrograms of VEGI-CTT were
applied into 12% SDS-PAGE, and then electrotransferred to a PVDF membrane.
Western blotting analysis was performed using mouse anti-His-Tag
monoclonal antibody (Lane 2). The membrane was incubated with the
second antibody: HRP-labeled goat antimouse IgG. Lane 1: prestained
molecular weight protein markers. Lane 2: fusion protein VEGI-CTT of
22 kDa.


## 3.3. Chimeric Protein VEGI-CTT Inhibits the
Proliferation of the Endothelial Cells in Vitro

The bioactivity of VEGI-CTT was determined
by the proliferation of ABAE and HUVE cells in vitro. As shown in Figure5(a), the
protein was found to preferentially inhibit the FGF-2-induced proliferation of
ABAE and HUVE cells in a dose-dependent manner, whereas it could not inhibit
the growth of HVSM, liver cancer HepG2 or NIH 3T3 cells. The half-maximum
inhibitory concentrations (IC50) of VEGI-CTT
for ABAE and HUVE cells were about 25 ng/mL and 100 ng/mL, respectively. The inhibition
effect of VEGI-CTT on endothelial cells was similar to that of VEGI alone or of
VEGI and CTT combined treatment at a concentration of 40 ng/mL. No effect on the
proliferation of the endothelial cells or cancer cells was observed in the
group of CTT treatment (Figure 5(b)). These
results suggest that recombinant protein VEGI-CTT, which is bioactive, could specifically
inhibit proliferation of endothelial cells when compared with other tested cell
types.Specific inhibition of endothelial cell proliferation by chimeric
protein VEGI-CTT. Cells were seeded in triplicate at
3000–5000 cells/well in 96-well plates. Culture conditions
are described in Section2. The media were changed once on day 3.
The number of viable cells was determined on day 6 by using a
Coulter counter. Mean values are presented as a function of VEGI-CTT
or other agents’ concentrations.  P*<.05 versus
that of PBS group.
(a)(b)


## 3.4. Inhibition of in Vitro Angiogenesis by
Chimeric Protein VEGI-CTT

The antiangiogenic activity of the recombinant VEGI-CTT
was examined with an in vitro angiogenesis model. In this model, when
recombinant VEGI-CTT was added to the cell cultures together with FGF-2,
inhibition of the formation of capillary-like tubes by ABAE cells was observed (Figure6). The relative intensities of the tubules were
determined by using computer-assisted image analysis. The IC50 value
for the inhibition was found to be approximately 40 ng/mL. VEGI used alone or VEGI and CTT combined treatment also could inhibit the formation
of capillary-like tubes by ABAE cells similar to those of VEGI-CTT, whereas
the antiangiogenic activity of CTT was not observed (Figure 6). These results further
demonstrated that recombinant protein VEGI-CTT had a potent activity of antiangiogenesis
through inhibiting the proliferation of endothelial cells.Figure 6
Inhibition of formation of capillary-like tubules by adult bovine
aortic endothelial (ABAE) cells in collagen gels. The experiments
were carried out in triplicate. At least 9 image areas were analyzed
for each VEGI concentration. Mean values and standard deviations are
presented as a function of VEGI concentration.P*<.05 versus that of PBS group.


## 3.5. VEGI-CTT Potently Reduced the Activity of MMP-2 and MMP-9

CTT is a recently described cyclic peptide collagenase inhibitor of MMP-2 and MMP-9. To determine
whether the recombinant protein we had expressed and renatured also could efficiently
inhibit the activity of MMP-2 and MMP-9, metalloproteinase inhibitory activity
of VEGI-CTT was assessed by casein degradation assay. In order to precisely
compare the bioactivity between recombinant protein VEGI-CTT and CTT peptide, molar
concentration was used here. As shown in Figure7, degradation of β-casein by MMP-9 or MMP-2 was inhibited by VEGI-CTT, similar
to that of CTT or both CTT and VEGI. Moreover, the inhibitory effect of
VEGI-CTT occurred in a dose-dependent
manner. For MMP-2, the fusion protein VEGI-CTT
inhibited MMP-2 with an IC50 of 150 ng/mL (equal to 7.5μM). Caseinolysis by MMP-9 was similarly inhibited by the protein
at high concentrations with an IC50 of 650 ng/mL (equal to 30μM). The differences of IC50 for MMP-2 and MMP-9
may be due to the different avidity of VEGI-CTT with them. However,
VEGI alone could not inhibit the degradation
of β-casein by MMP-9 or MMP-2. The data indicated that recombinant
protein VEGI-CTT had similar activity to the CTT peptide as previously reported,
and could potently
reduce the activity of MMP-2 and MMP-9 in a dose-dependent manner.Inhibition of caseinolytic activity by the chimeric protein
VEGI-CTT. MMP-2  and MMP-9 enzymes were incubated with 2 mg/mLβ-casein in
the absence or presence of VEGI-CTT. Proteins were separated on a 10% SDS-polyacrylamide
gel, and detected with Serve blue R. For details, see Section 2.
Inhibition of MMP-2 and MMP-9 mediated β-casein degradation by
VEGI-CTT at different concentrations with apparent IC50 of 7.5μM
and 30μM, respectively.
(a)(b)


## 3.6. Xenograft Antitumor Activity

We extended our analysis of the antitumor activity of chimeric
protein VEGI-CTT using mouse xenograft models. Cultured CA46 lymphoma cells
were implanted subcutaneously into nude mice, the agent treatment started upon tumor
xenograft development, and the tumor growth was monitored for 2 weeks.
Subcutaneous inoculation of1×107 CA46 cells resulted in
aggressively growing tumors in PBS treatment group (Figure 8). Tumor-bearing mice
were treated with predetermined doses of chimeric protein VEGI-CTT, and
significant reduction in tumor burden was observed. Treatment with 50 ug/mouse VEGI-CTT
twice a week reduced tumor growth by 82.2% compared with animals in the PBS-treated
group. In contrast, treatment with 50 ug/mouse VEGI or CTT peptide alone merely
resulted in 49.2% and 35.1% reduction, respectively. In addition, compared with
VEGI-CTT treatment, combined treatment with 50 ug/mouse VEGI and CTT peptide had
a relatively low antitumor activity but also significantly reduced tumor growth
by 62.9%. These results establish chimeric protein VEGI-CTT as an effective
inhibitor of lymphoma tumor growth in vivo, and thus it had a more potent
antitumor activity than VEGI, CTT, or the combination of both.Figure 8
Effect of chimeric protein VEGI-CTT in treating CA46 human
lymphoma xenograft-bearing nude mice. Nude mice were inoculated with
CA46 human lymphoma cells, and the treatment started upon tumor
xenograft establishment in nude mice. Tumor xenograft-bearing nude
mice were injected with indicated doses of VEGI-CTT, VEGI, CTT, or
VEGI and CTT combined treatment for two weeks, and tumor volumes
were measured on alternate days. Mean values and standard deviations
are presented as tumor volumes in each group (n=9). The experiments
were repeated with similar results.


## 4. Discussion

Angiogenesis is required for tumor growth and metastasis,
and its inhibition may be a new valuable approach to cancer therapy. It has
been demonstrated that a variety of endogenous angiogenesis inhibitors such as
angiostatin [16], endostatin [17], restin [18, 19], canstatin [20, 21], and
tumstatin [22, 23] can lead to the suppression of primary and metastatic tumors’
growth without affecting the normal vasculature growth. Advantages of antiangiogenic
therapy include easy access of drugs to the endothelial cell compartment and
low drug resistance. Vascular endothelial cell growth inhibitor (VEGI), a novel
cytokine which belongs to the TNF superfamily, is one of the most potent
inhibitors of endothelial cell proliferation and migration as well as tumor
growth in mice [4–6]. The synthetic CTTHWGFTLC-deca peptide (CTT peptide) has
been recently described as a specific and selective inhibitor for MMP-2 and -9.
CTT peptide has been shown to suppress invasion and migration of both tumor and
endothelial cells in vitro, prevent the growth and invasion of tumors in mice,
and specifically home to tumor vasculature expressing gelatinases (MMP-2 and
-9) in vivo [13, 14].In the present study, we incorporated the extracellular
domain of VEGI with CTTHWGFTLC-deca peptide for antitumor therapy by more
potent antiangiogenesis. The fusion protein VEGI-CTT was expressed inE. coli. Recombinant proteins expressed
in bacteria often form inclusion bodies, especially when they are expressed at
high levels [24]. In our case, the expressed protein formed inclusion bodies.
Therefore, an efficient and convenient refolding system that would help the
purification of VEGI-CTT is needed [25]. Renaturation of VEGI-CTT is very
crucial because it is closely related with the final yields. There are many
methods for refolding, such as dilution, dialysis, and gel filtration [26–28]. In this
work, we chose on-column metal ion-affinity chromatography for protein
refolding. The purity of inclusion bodies is a key factor. Previous reports
demonstrate that the washing step can remove contaminants, especially proteins
that may have been absorbed
onto the hydrophobic inclusion bodies during processing and could affect
protein-refolding yield [29]. In the present study, inclusion bodies were
washed three times. Washing with 2 M urea using 1%
Triton X-100 effectively removed remaining cellular proteins. The final purity
of the inclusion bodies could reach 80% after the last washing, facilitating
the following purification and refolding. Another important factor to influence
the yield of refolding is the time and flow rate. We tested three different
times (1, 2, and 4 hours) and three different flow rates (2, 1, and 0.5 mL/min) for on-column
refolding. The result showed that there was a lot of precipitation at 2 mL/min
(1, 2, and 4 hours), with some at 1 mL/min (1 and 2 hours) and 0.5 mL/min (1 hour)
and little at 0.5 mL/min (4 hours). We chose 0.5 mL/min (4 hours) as the final flow
rate for refolding process. This provided the opportunity to generate more
soluble refolded VEGI-CTT and increase the yield of the protein. In our study,
we use Ni2+-chelating column to refold, purify, and reform disulfide
bonds of VEGI-CTT in one step. By this strategy, an amount of the obtained refolded
recombinant VEGI-CTT was approximately 25.5 mg/L of initial bacterial broth. The
results indicate that this process for renaturation and purification of VEGI-CTT
is suitable and effective.Our purpose was to express and purify VEGI-CTT, and use
active VEGI-CTT to obtain more potent antitumor effect than that of VEGI or CTT
peptide alone. So we confirmed the bioactivities of VEGI-CTT by a series of
assays in vitro. The results showed that VEGI-CTT has both activities exerted
by VEGI or CTT alone. VEGI-CTT could effectively inhibit endothelial cell
proliferation and the formation of capillary-like tubes by ABAE cells; meanwhile,
VEGI-CTT could potently reduce the activity of MMP-2 and MMP-9. Importantly, the
further
in vivo study demonstrated that chimeric protein VEGI-CTT has more potent
antitumor activity not only than that of VEGI or CTT, but also than that of the addition of VEGI
and CTT in combination. The explanation may be that chimeric protein VEGI-CTT
can more efficiently
increase the half-life of CTT than CTT peptide used alone in vivo. Further studies with recombinant VEGI-CTT are underway to
test the ability to inhibit tumor invasion or metastasis in vivo, which may
give insight into its unique molecular mechanisms underlying the antitumor
actions of VEGI-CTT.



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*Source: 564969-2008-08-20.xml*