| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
University of Pittsburgh Cancer Institute and Department of Pathology [X. H., Q. Z., Z. Z., K. Z. Q. W., N. H., C. Y., E. G., M. L.] and Department of Medicine, Division of Hematology-Oncology [M. K. K. W.], University of Pittsburgh, Pittsburgh, Pennsylvania 15213
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Endostatin is a Mr 20,000 protein that specifically and strongly inhibits tumor angiogenesis. It was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line and represents a COOH-terminal fragment of collagen XVIII. The endostatin gene has been cloned and expressed as a recombinant protein in Escherichia coli and yeast expression systems (2 , 3) . Animal studies demonstrated that recombinant endostatin strongly inhibits the growth of a variety of murine and xenotransplanted human tumors (4 , 5) .
Purification of bioactive recombinant endostatin from E. coli, however, has proved challenging because the endostatin is purified under denaturing condition (e.g., urea) but invariably becomes insoluble under physiological conditions. The most likely biochemical explanation for this precipitation phenomenon is that endostatin becomes misfolded during the process of renaturation. It is believed that injection of precipitated endostatin into animal results in in vivo conversion into a soluble form, thus enabling it to exert its antiangiogenic and antitumor activities (2) . However, this refolding process likely depends on a variety of in vivo factors that are biologically variable, thus rendering this in vivo renaturation unpredictable. In addition, accurately assaying insoluble endostatin is problematic. For this and other reasons, the efficacy and activity of endostatin are difficult to reproduce accurately. Because of these issues, insoluble endostatin is unlikely to become clinically useful.
In recognition of the shortcomings of insoluble endostatin, a soluble form of recombinant endostatin was produced via a yeast expression system (3) . However, the relatively low yield of the system has made it difficult to produce this agent in quantities sufficient for extensive clinical evaluation. In addition, its production is very expensive and represents a large step up in cost over bacterial recombinant methods. These barriers have hampered the widespread translation of endostatin research to clinical practice. There is therefore a great need to increase the yield and to reduce the cost of the production of recombinant endostatin that is suitable for clinical use. Our strategy centered on the optimization of the E. coli expression system because of its higher efficiency in expressing foreign proteins as compared with the yeast system. In this study, we demonstrate generation of soluble recombinant murine endostatin from E. coli and evaluate its antiangiogenic and antitumor activities. We have developed a purification protocol by which recombinant endostatin can be expressed with high efficiency, purified as inclusion bodies, and finally refolded into a soluble conformation. Our in vivo experiments showed that the SRE3 possesses antiangiogenic and antitumor activity comparable with the originally published insoluble form.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
For small-scale expression screening, pTB01#4 was introduced into competent BL21(DE3)pLysS bacterial cells as described by the manufacturer. Ten colonies were randomly picked from the obtained transformants on a Kan+LB plate (10 grams/liter tryptone, 5 grams/liter yeast extract, 10 grams/liter NaCl, and 50 mg/liter kanamycin), and each of these was inoculated into 1 ml of Luria-Bertani medium containing 50 mg/liter kanamycin. These 10 inoculates were cultured in a 37°C shaker overnight at 280 rpm, and 50 µl of bacteria from each overnight culture were then transferred to a new tube containing 1 ml of the same broth, followed by continuous shaking culture at 37°C until A600 nm reached 0.8. At this point, IPTG was added to the culture at a final concentration of 0.3 mM to induce the expression of endostatin. After culturing in a 37°C shaker for another 3-h period, 10 µl of bacteria sample were collected from each 1-ml culture, followed by microcentrifugation at 1000 rpm for 5 min at 4°C. The bacterial pellets were resuspended with 10 µl of 1x sampling buffer [50 m[scap]m Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromphenol blue, and 10% glycerol] and analyzed by SDS-PAGE.
The clone with the highest expression efficiency was identified from the 1-ml expression screening experiment by SDS-PAGE and subsequently applied to a 1-liter scale expression. The culture and induction condition for 1-liter scale expression were the same as that used for the 1-ml culture modified by simply scaling up each component by 1000 times.
Purification of Recombinant Endostatin from E.
coli in an Insoluble Form.
To evaluate the biological activity of the soluble endostatin produced
in this study, we used IRE as a reference for comparative studies. A
purification procedure for recombinant endostatin from E.
coli has been described previously (2)
. Briefly,
bacteria pellet was collected with low-speed centrifugation, followed
by lysis with 8 M urea. The lysate was then
applied to a Ni2-NTA column (Qiagen). After
washing with 8 M urea containing 10
mM imidazole, endostatin was eluted with 8
M urea containing 250 mM
imidazole. The dialysis product was subject to an endotoxin level
determination (Limulu Amebocyte Lysate Progent/plus; Biowhittaker,
Inc., Walkersville, MD). Quantification of the endostatin protein
before dialysis was performed using the Bio-Rad protein dye method as
described by the manufacturer. Finally, the endostatin product was
dialyzed against 1x PBS (molecular weight cutoff, 6000–8000)
at 4°C. During the dialysis, the purified protein precipitates to
form IRE.
Purification of SRE from E. coli.
Bacteria were collected from the IPTG-induced 1-liter culture by
centrifugation at 2500 x g for 10 min at
4°C. The pellet was then resuspended in 100 ml of buffer A [0.1
M Tris-HCl (pH 8.0) and 5
mM EDTA], followed by incubation at room
temperature for 15 min, with the addition of lysozyme at a final
concentration of 50 µg/ml. The suspension was then sonicated with a
VibraCell VC50 sonicator (Sonic & Materials, Inc.) in the presence of
0.1% sodium deoxycholate, followed by centrifugation at 8000 x g for 10 min. The supernatant was discarded, and the
pellet was resuspended in 100 ml of buffer A containing 0.1% sodium
deoxycholate. The centrifugation/resuspension procedure was repeated
twice. The resultant pellet from the last centrifugation was dissolved
in 30 ml of buffer B [0.05 M Tris (pH 8.0), 1%
SLS, and 1 mM DTT] and centrifuged at
8000 x g for 10 min at 4°C. The clear
supernatant obtained was then transferred to dialysis tubing with a
molecular weight cutoff of 8000 and dialyzed twice against 1500 ml of
buffer C [0.05 M Tris-HCl, (pH 8.0) and 0.1
mM DTT] at 4°C for 4 h. The recombinant
protein was then further dialyzed twice against 1500 ml of buffer D
[0.05 M Tris-HCl (pH 8.0)] and 1000 ml of
buffer E [0.05 M Tris-HCl (pH 8.0), 0.01
mM oxidized glutathione, and 1
mM reduced glutathione] at 4°C for 4
h/dialysis cycle, respectively. A final dialysis (4°C, 5 h)
against 0.05 M Tris-HCl (pH 8.0) was performed
twice to eliminate the glutathione redox pair. The dialysis product was
subject to endotoxin level determination. The purified protein was
finally quantitated by the Bio-Rad protein dye method as described by
the manufacturer, aliquoted, and stored at -20°C.
SDS-PAGE Analysis.
SDS-PAGE was performed according to a standard procedure under reducing
condition (9)
. Bacterial lysates from the small-scale
expression screening procedure and 10 µg of SRE obtained as the final
purification product were used as SDS-PAGE samples, respectively.
Briefly, samples suspended in 10 µl of 1x sampling buffer were
loaded on a 15% SDS-PAGE gel and run until the prestained molecular
weight standard (Bio-Rad) revealed a good resolution, followed by
staining with Coomassie Blue and destaining with methanol and acetic
acid.
Measurement of Residual Detergent (SLS) in Purified SRE.
The SLS concentration in purified SRE was determined using a
turbidimetric assay (10)
. Briefly, the SRE sample was
serially diluted in distilled water to a final volume of 1 ml, and then
0.1 ml of 1 M HCl was added to each dilution and mixed by
vortexing. After a 5-min incubation at room temperature, the
A450 nm values of the mixtures were
measured. The amount of SLS present in the SRE sample was calculated
from a standard curve made by using a series SLS with known
concentrations ranging from 0.0025–0.025%. In this method, the
A450 nm value is a linear function of
SLS concentration between 0.005% (0.05 mg/ml) and 0.05% (0.5 mg/ml).
Testing of the Antiangiogenic Effect of Recombinant Endostatin.
In vivo Matrigel assay was used to test the antiangiogenic
effect of soluble endostatin as described previously (11)
.
Phenol red-free, growth factorreduced Matrigel (Collaborative
Biomed and Cell Products, Bedford, MA) was mixed with 50 ng/ml
recombinant human FGF-1 prepared as described previously
(12)
and 1 unit/ml heparin (Elkins-Sinn, Cherry Hill, NJ).
C57BL/6 mice received s.c. injection in each flank of 0.4 ml of the
prepared Matrigel. One day after injection, mice were divided into
three groups (five mice/group). A control group was treated s.c. with
PBS, and the rest of the groups were treated s.c. with either SRE or
IRE (20 mg/kg, twice a day). Endostatin was injected at sites distant
from the site of the Matrigel injection. Mice were sacrificed
after 10 days of treatment. Harvested Matrigel plugs were fixed
overnight in 10% buffered formalin at 4°C and then placed into 70%
ethanol before being processed for paraffin sections.
Immunohistochemical staining for factor VIII was carried out using
Vectastain ABC according to the manufacturer’s instructions. Primary
rabbit-derived antibody to human factor VIII was obtained from Dako.
All photomicrographs were taken under identical conditions.
Confluent cultures of human umbilical vein endothelial cells were used to assess the ability of endostatin to inhibit endothelial cell migration and growth. These cells were grown with DMEM/10% FCS on 60-mm Falcon tissue culture dishes. Once the culture became confluent, half of the cell monolayer was sharply denuded using a sterile razor, and the demarcation line was etched onto the tissue culture vessel. The culture was washed with sterile PBS and incubated in DMEM/10% FCS/FGF-1 (10 ng/ml) containing either 0, 5, 10, or 30 ng/ml SRE. Cells were fed daily and fixed with 5% buffered formaldehyde 96 h after monolayer denudation. A calibrated ocular micrometer measured the distance traveled by the endothelial monolayer from the original denudation line. Measurements were taken from a point 0.5 cm proximal and distal from the center of the dish. Three cultures were used to test each concentration of endostatin.
In Vivo Evaluation of the Antitumor Effect of
Recombinant Endostatin.
The 3LL Lewis lung carcinoma model has been used extensively in
preclinical testing of endostatin (2
, 4
, 5
, 13)
. In this
study, we used 3LL-C75, a cell clone isolated from 3LL Lewis lung
carcinoma irradiated with UV light (14)
, to assess the
antitumor effect of recombinant endostatin. In a separate set of animal
experiments performed in our laboratory, this clone exhibited higher
sensitivity to the growth-inhibitory effect of endostatin than the
original 3LL
tumor.4
Tumor cells were cultured in RPMI 1640 supplemented with 10% FCS, 2
mM glutamine, and antibiotics. 3LL-C75 cells (1 x 106) were inoculated into C57BL/6 mice.
When tumors reached about 0.5 cm in diameter, mice were divided into
three groups (five mice/group). Two groups of mice were treated s.c.
with either SRE or IRE (20 mg/kg) twice a day. The control group
received PBS injections. Tumor size was monitored by measuring the
longest dimension (length) and shortest dimension three times/week with
a dial caliper, and the tumor volume was calculated as
width2 x length x 0.52
(15)
. All data are presented as mean ± SE.
The experiments were terminated when tumors in control groups reached
2.0 cm in diameter or induced undue morbidity as per the protocol
approved by the University of Pittsburgh Institutional Animal Care and
Use Committee.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
In our protocol, the yield of purified SRE is approximately 150
mg/liter culture. This yield is similar to the IRE production made in
our laboratory with the published insoluble protocol (2)
.
The final dialysate in the soluble protocol did not contain any visible
precipitants or fine particles and remained solubilized either at 4°C
or after melting from -20°C storage. The levels of endotoxin in
purified SRE and IRE were lower than 8 endotoxin unit/ml. When
10 µg of protein were analyzed by reducing SDS-PAGE, a single band of
Mr 20,000 corresponding to endostatin
was seen (Fig. 1)
. The identity of this homogeneous preparation was further confirmed by
an endostatin-specific ELISA test (Cytimmune Science, College Park,
MD). Almost all of the protein is reactive to the antiendostatin
antibody provided in the kit (data not shown). Turbidimetric assessment
of SLS showed that after serial dialysis, the amount of residual
detergent in our final product was less than 0.005% (0.05 mg/ml),
indicating that more than 99.5% of SLS used to solubilize the
inclusion bodies was removed. The solubility remains stable even after
the redox pair is eliminated from the system, indicating that this
approach is effective in making soluble recombinant murine endostatin.
|
. In marked contrast, there is a paucity
of cells within Matrigel harvested from mice treated with insoluble
(Fig. 2C)
or soluble (Fig. 2D)
endostatin (20
mg/kg/day, 9 days). No inflammation was evident in any of these
sections. These results indicate that at this dose level, the
antiangiogenic effects of SRE and IRE were equivalently potent.
|
In Vivo Antitumor Effect of SRE.
To assess the antitumor activity of the obtained SRE, we used the
3LL-C75 lung carcinoma model in this study. C57BL/6 mice bearing a
tumor of about 0.5 cm in diameter were divided into three groups (five
mice/group). Two groups of mice were treated twice with 20 mg/kg/day of
either SRE or IRE. Control mice received injection of PBS in parallel.
A substantial inhibitory effect was observed in mice treated with
either IRE or SRE, and the degree of inhibition appeared to be similar
(Fig. 3)
. These results suggest that at this dose, the bioactivity of the
soluble form of recombinant endostatin is similar to that of insoluble
endostatin. Thus, an expression and purification system for SRE
protein from E. coli has been successfully
established in a laboratory setting.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The IPTG-inducible T7lac promoter used in our system has previously been shown to be highly efficient in expressing heterologous proteins, including endostatin (2 , 16) . With the conventional purification protocol described previously (2) , the cultured bacteria are lysed under denaturing condition (8 M urea), and the dissolved endostatin is then subject to affinity chromatography for His tag-specific purification. However, the purified protein misfolds and precipitates during the dialysis that eliminates urea from the solvent system (2) . In this study, we used a different purification approach that takes the advantages of inclusion body formation in the endostatin-expressing E. coli cells. Many proteins expressed in the described system aggregate and form intrabacterial inclusion bodies. The inclusion bodies can be easily collected by simple procedures such as centrifugation so that the target protein can be highly enriched at early stages of purification. The enrichment, followed by a rinsing procedure, could virtually be optimized to achieve high purity without further purification procedures such as affinity chromatography. In addition, inclusion bodies also provide the aggregated protein molecules with protection against degradation by intracellular proteases, which is not an uncommon posttranslational event for proteins heterologously expressed in bacteria. In most cases, however, protein molecules aggregated in inclusion bodies are not correctly folded, resulting in the generation of insoluble and/or biologically inactive molecules. Therefore, recovery of a recombinant protein from inclusion bodies requires an effective refolding process with optimized conditions that might vary from protein to protein. In our approach, detergent SLS and reducing agent DTT were used to dissolve the inclusion bodies that are partly but highly purified by repeated rinsing with a mild detergent-containing buffer. The dissolved endostatin molecules are then subject to a refolding procedure using a redox pair and oxidized and reduced glutathiones to facilitate formation of a stable conformation that makes and keeps the endostatin soluble.
This study outlines a strategy for the isolation of a soluble form of endostatin. We have focused this strategy on exhibiting that SRE is equivalent to IRE at a typical dose level (20 mg/kg/12 h) previously shown to be active in a large number of studies (4 , 13) . The true potency of SRE will require more extensive dose ranging studies. However, our preliminary data show that the same dose of SRE given once daily gives a similar angiogenesis inhibition as shown here (data not shown). Thus, we anticipate that this soluble material will have potency over a large dose range.
Endostatin is a potent antiangiogenic protein and an antitumor factor. However, the insoluble nature of the published version of E. coli recombinant endostatin hampered its clinical application. Although soluble endostatin prepared from a yeast system is being used in ongoing Phase I clinical trials, the low yield (approximately 20 mg/liter culture) and high cost of the system have made it difficult to produce in quantities that are realistic for comprehensive clinical evaluation and application. Our results presented in this report offer an alternative method that will prove valuable in helping to determine the clinical activity of endostatin. Obviously, it will be of great interest and importance to adopt a similar method in preparation of soluble human endostatin. This subject is being explored in our laboratory.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by American Cancer Society Grant
IRG-60-002-39. 
2 To whom requests for reprints should be
addressed, at University of Pittsburgh Cancer Institute, Biomedical
Science Tower, Room W1053, 200 Lothrop Street, Pittsburgh, PA 15213.
Phone: (412) 624-1490; Fax: (412) 624-7736; E-mail: mengfeng{at}pitt.edu
3 The abbreviations used are: SRE, soluble
recombinant endostatin; IPTG,
isopropyl-1-thio-ß-D-galactopyranoside; IRE, insoluble
recombinant endostatin; FGF, fibroblast growth factor; SLS,sodium N-laurylsarcosine.
Received 7/19/00. Accepted 11/29/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  F. Ou-Yang, K.-L. Lan, C.-T. Chen, J.-C. Liu, C.-L. Weng, C.-K. Chou, X. Xie, J.-Y. Hung, Y. Wei, G. N. Hortobagyi, et al. Endostatin-Cytosine Deaminase Fusion Protein Suppresses Tumor Growth by Targeting Neovascular Endothelial Cells Cancer Res., January 1, 2006; 66(1): 378 - 384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, W. Wang, and Y. Luo Contributions of Disulfide Bonds in a Nested Pattern to the Structure, Stability, and Biological Functions of Endostatin J. Biol. Chem., March 25, 2005; 280(12): 11303 - 11312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Li, C. Ye, C. Feng, F. Riedel, X. Liu, Q. Zeng, and J. R. Grandis Enhanced Antiangiogenic Therapy of Squamous Cell Carcinoma by Combined Endostatin and Epidermal Growth Factor Receptor-Antisense Therapy Clin. Cancer Res., November 1, 2002; 8(11): 3570 - 3578. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ye, C. Feng, S. Wang, X. Liu, Y. Lin, and M. Li Antiangiogenic and Antitumor Effects of Endostatin on Follicular Thyroid Carcinoma Endocrinology, September 1, 2002; 143(9): 3522 - 3528. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) treatment was performed by s.c. injection of 20
mg/kg/12 h, respectively. Control mice (
) were treated with PBS in
parallel. Tumor volumes were measured as described in "Materials and
Methods."