I.
Introduction
Tumstatin
is a ramification of basement
membrane proteins
in human body (28Kda, an endogenously produced a third a chain of basement membrane
collagen, type IV). It inhibited specific for the protein
synthesis of endothelial cells (Maeshima et al, 2002). In the
experiment on rats, it showed that Tumstatin could inhibit tumor growth (Maeshima et al, 2000),
and anti-tumor activity of Tumstatin was also verified (Maeshima et al, 2002).
A physician, J. Folkman in Harvard medical collage in USA firstly mentioned the
theory inhibiting tumors through angiogenesis. He thought that if the blood
vessels of tumors were inhibited, tumors could not get hyperplasia, metastasis
instead of shrinking. Tumstatin
prevents angiogenesis through inhibition of endothelial cell proliferation and
promotion of apoptosis with no effect on migration, whereas endostatin prevents
endothelial cell migration with no effect on proliferation. Therefore,
it probably fit for curing many types of cancers. Because of the distinct
properties of tumstatin and endostatin, it indicated that they had diverse
antiangiogenic actions (Sudhakaret al 2003).
Up to now,
few of the structure, characteristics and its¡¯ protein knowledge of tumstatin
has been known, and in particular, the report on the gene of tumstatin from Chinese
human tissues has not been found yet. Additionally, Purification
of bioactive recombinant protein from E. coli has been recognized
challenging. ¡¡Our strategy
would center on the optimization of the E. coli expression system
because of its higher efficiency in expressing foreign proteins as
compared with the other systems. The study showed the cloning,
expression, purification and its¡¯ activity of tumstatin from Chinese kidney
tissues. It would lay a theoretical foundation for the clinical application on
tumstatin.
II.
Materials and methods
A.
Material, bacterial strains and reagents
The
kidney tissue of abortus fetus were collected from associated hospital in
medical college Inner Mongolia. PET-His expressive vector, E.coli host
strain, DH5¦Á, and BL21(DE3)plysS stored in our laboratory, RNA purified kit (Shanghai Huashun
Co), pGEM-Tvector kit, T4 DNA ligase and plasmid purified kit (Promega), restriction enzyme, BamH I, Nhe
I (NEB), Taq plus DNA polymerase, dNTPs, X-gal, IPTG and agarose( biotechnology Co,¡¡ Shanghai), DL2000 DNA
molecular weight marker and multi clone antibody(invitrogen), HRP-labaled IgG
of sheep against mouse(Huamei Co in Beijing).
B. Combined buffer
20
mmol/L NaH2PO4, 500mmol/L NaCI, pH 7.4, Washing buffer NaH2PO420
mmol/L, NaCI 500 mmol/L, imidazole 500 mmol/L, pH 7.4.
C. Sequencing and cloning of Tumstatin
1.
Synthesis and designing of the primers
A
pair of primers was designed according to the sequence from GenBank (No. AF258351),
tum1: 5¢-CGGGATCCCCAGGTTTGAAAGG-3¢and tum2: 5¢-GGCTAGCGTGTCTTTTCTTCATGCACA-3¢, underlined nucleotides
indicated the recognized sites of restriction enzyme as BamHI, NheI.
Amplified the fragment of gene was about 750 nt long.
2. Preparation of template
Total
RNA of kidney from Chinese abortus fetus was isolated with RNA extract kit.
Reverse transcription (RT) would carried out when the content and purity were
qualified. It did according to the instruction of the RT kit. PCR would be done
with the cDNA synthesized as a template.
3 Cloning and sequencing of Tumstatin
SuperScriptTM
First-Strand Synthesis System for RT-PCR (Invitrogen) E.coli Top10 was grown on Luria-Bertani (LB) medium and incubated at 37¡æ under
aeration. Amplification reactions were performed in a total volume of 50 ml containing 100 ¦ÌM (each) dATP, dCTP, dGTP, and dTTP, 25 pmol
of each primer, 2 ng of pLSC400 DNA, 2.5 U of Pwo DNA
polymerase (Boehringer, Mannheim, Germany), and the corresponding 1¡Á
Pwo buffer. Reactions were carried out with a Perkin-Elmer
thermocycler by using initial denaturation at 94¡ãC for 5 min,
followed by 5 cycles consisting of 94¡ãC for 30s, 46¡ãC for 30s, and
72¡ãC for 80s and followed by 25 cycles consisting of 94¡ãC
for 30s, 55¡ãC for 30s, and 72¡ãC for 80s a final extension step
consisting of 72¡ãC for 10 min. The amplified products were
identified by electrophorsis of 1% agarose. Each DNA was further purified by
treatment with phenol-chloroform as described by Sambrook et al, 1989. Plasmid DNA was
isolated from the recombinant E.coli by a method described previously
(Sambrook et al, 1989). DNA sequences were determined by the dideoxy chain
termination method with sequencing kits (Biotechnology Co, Shanghai). The purpose
product by PCR was ligated with GEM-T vector, then transformed to E.coli
DH5¦Á competent cells by the method of CaCl2. And recombinant were
selected through blue and white spots, and identified by situ-PCR and
endoenzyme digesting. The positive recombinant plasmid would be sequenced by
Biotechnology Co, Shanghai.
D. Construction and inducing expression of
pET-His-tumstatin plasmid
The
extracted plasmid containing tumstatin gene, pGEM-T/tum was digested with BamHI and NheI. Tumstatin DNA was
recollected and cloned into expressive vector, pET-His digested with the same
two enzymes, that contained an NcoI site and a PstI recognition sequence within
the forward and reverse primers, respectively. The amplified product
was digested with NcoI and PstI and cloned into expressive vector, pET-His
digested with the same two enzymes mentioned above and ligated to generate
plasmids pET-His-tumstatin. The plasmids were subsequently transferred to
E coli cells. The recombinants were selected and identified named
pET-His-tumstatin. The pET-His-tumstatin were also transferred to E.coli
BL21 (DE3) plysS competent cells. and¡¡
the positive bacteria were identified by PCR. The bacteria selected were
incubated in LB medium induced with IPTG in different concentrations
of 0.2, 0.4, 0.6, 0.8 mmol/L at the same time. And the bacteria were sampled
0.2ml once each before and after inducing. The samples were precipitated and cellular
proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The quantity of expression was analyzed by Gel imaging
instrument made in Japan.
E. Tumstatin purification
To determine Tumstatin activities, the engineering E.coli added
in 1000 ml of LB culture with a 1/100 volume. And E.coli was grown at
37¡ãC to an optical density of 0.5 at 570
nm. The culture was added with a final concentration of 0.2 mmol/L IPTG
at 37¡ãC for 3h. The cell culture were pelleted by centrifugation at 5000 ¡Á g
for 10 min, and the cells were resuspended in 100ml of 30mM PBS buffer, and
centrifugation at 5000 ¡Á g for 10 min, and resuspended as
above mentioned. Cell lysis was carried out by ultrosonic way. And the cell
fragments were removed by centrifugation at 13,000 ¡Á g for
30 min. The supernatant is run through a volume of 5 ml
HiTrap chelating Ni-NTA column (Amersham Pharmacia). ÄKTA FPLC purifying system for protein would be connected
with the column. The column is
then washed with the washing
buffer, followed by elution of the bound protein from the column using the
elution buffer. Finally, the column is re-equilibrated with washing buffer. washing or eluting, the
compounds down the column by varying the eluting solvent using
a flow rate of 1ml/min. And all the fractions were pooled
with an absorbance > ~0.03. And the tumstatin solution
was concentrated using Ultrafree-15 concentrators of 10kDa. The Ultrafree-15 concentrators are used to concentrate protein
samples based on a technique known as ultrafiltration. These disposable devices
hold up to 15 ml of sample at a time and can be centrifuged at 2,000 ¡Á g
for 15 min, and the step was repeated for 3 times (refer to the
Ultrafree-15 manual for more information). The samples were pooled and resolved with sterilized PBS of 10ml. The samples were
analyzed by the gel of 15 % SDS-PAGE and also quantified with the method
of Bradford.
F. Identification of Tumstatin
1. Western
blot of tumstatin
After running SDS-PAGE (Sambrook et al, 1989), the extracts were
transferred to nitrocellulose membrane (Sigma). Blots were stained
firstly with Ponceau dye for 2 min and then developed with
first antibody (antiserum against V5 from mice), followed by staining with
secondary antibody (horseradish peroxidase labeled anti-mouse IgG).
2.
Detection using indirect ELISA
For visualization, nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate was used. Tumstatin
(450 mg/ml) were diluted at 1:100, 1:200, 1:400 and 1:800
respectively, and added 100 ml each hole on plate of 96
holes at 4¡æ overnight. Next day, the plate was blocked with
3% bovine serum albumin in Tris buffered saline with 0.1% Tween 20
for 1 h and incubated with 100 ml first antibody (polyclone
against-mouse antiserum)at 1:500 dilutions for 30min at 37¡æ. After washing with
PBST, it also was incubated with 100 ml of HRP labeled anti-mouse
IgG at 1:1000 dilutions and washed as above mentioned. A drop of
developing fluid A and B were added respectively for 5min followed by adding a
drop of terminal reactive fluid. OD values were determined at 450nm.The
positive was determined according to the ratio of experimental holes to
negative holes if the ratios were larger than 2.1.
3. Antitumor effect of Tumstatin
Twenty 7-week-old male Kun Ming mice without thymus gland per group were
used as test animals. And kidney tumor induction was performed as follows. 786-0 nephrosarcoma cells were subcutaneously transplanted to the back
region, and attacking numbers of nephrosarcoma cell
were 2´106. After a week of injections, when the tumor growth volume was up to 600-700mm3, the ten mice were injected
tumstatin of 6 mg/kg subcutaneously
in the back region a time, and once a day for ten time injections. however, the another ten mice were only injected with
0.9% normal saline (vehicle) at the same time. Tumor growth volume
(width ´length ´0.52) needed to be determined with vernier caliper on a daily basis.
III. Results
A. Cloning and sequencing of tumstatin
PCR product of tumstatin would
run electrophoresis using 1%
agarose, the result showed the 700bp fragment presenting, and it was the same
as anticipation. It found the nucleotides of the 96th was mutated T®C but nonsense mutation (Figure 1).
B. Construction of expression vector
The fragment of
pGEM-T/tum-digested with BamHI and NheI was cloned into
pET-His plasmid, and ligation
product were transformed DH5¦Á competent cells. Thus 5 monoclonal colony were selected and identified by
PCR. DNA of positive plasmid was extracted and digested with BamHI and NheI.
An objective fragment of 741 bp was finally identified (Figure 2).
C. Expression and induction of recombinant
tumstatin
To investigate the regulation
of tumstatin expression
by IPTG and time, recombinant E.coli was each grown in three batches
by shaking conditions for approximately 3h. Through several conditions
obtimization, concentrations of IPTG added was 0.2 mmol/L IPTG and inducing
time was 3h at 30¡æ. The quantity of expression recombinant protein in gross
protein was about 35.66. It also presented either inclusion bodies or soluble
proteins (Figure 3).
Figure
1. Electrophoresis of PCR. 1.DL2000 DNA Marker, 2. PCR product of tumstatin, 3. negative control.
Figure
2. Vector pET-His-tumstatin digested with BamHI and NheI 1. DL2000
DNA Marker, 2. pET-His-tumstatin/
BamHI and NheI, 3.
pET-His-tumstatin, 4. pET-His.
