Original Paper
|
|
|
file on Synergy |
Acta Biochim Biophys
Sin 2006, 38: 305-309
doi:10.1111/j.1745-7270.2006.00167.x
Production of the Polyclonal Anti-human
Metallothionein 2A Antibody with Recombinant Protein Technology
Faiz m. m. t. Marikar, Qi-Ming Sun, and Zi-Chun Hua*
The State Key Laboratory
of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China
Received: February
12, 2006
Accepted: March 13,
2006
This work was
supported by the grants from the Ministry of Education of China (TRAPOYT 1999028418,
SRFDP 20030284040), and the National Nature Science Foundation of China (No.
30270291, 30330530, 20373026 and 30425009)
*Corresponding
author: Tel, 86-25-83324605; Fax, 86-25-83324605; E-mail, [email protected]
Abstract Metallothionein 2A (MT2A) is a small stress response
protein that can be induced by exposure to toxic metals. It is highly expressed
in breast cancer cells. In this study, the cDNA encoding the human MT2A protein
was expressed as glutathione S-transferase (GST) fusion protein in Escherichia
coli. Recombinant MT2A proteins were loaded onto 12% sodium
dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the
recombinant protein was visualized by Coomassie blue staining and the 33 kDa
recombinant GST-MT2A fusion protein band was cut out from the gel. The gel
slice was minced and used to generate polyclonal antisera. Immunization of
rabbit against MT2A protein allowed the production of high titer polyclonal
antiserum. This new polyclonal antibody recognized recombinant MT2A protein in western blot analysis. This low-cost
antibody will be useful for detection in various immuno-assays.
Key words metallothionein 2A; glutathione S-transferase; polyclonal
antibody
Genes encoding metallothionein, often in multiple copies, are found
in all eukaryote cells as well as some prokaryote cells [1]. Metallothioneins
are unusually rich in cysteine residues that coordinate multiple zinc and
copper ions under physiological conditions. The human metallothionein 2A (MT2A)
gene is expressed at all stages of development in many types of cells in most
organs, and is often coordinately regulated by toxic metals [2].Metallothionein is a small protein with a molecular weight of
approximately 6 kDa [3]. Several reports have indicated that there is an
enhanced expression of MT2A in primary breast carcinoma cells, and that it is
related to poor prognostic outcome [4]. MT2A expression has been considered to
be a useful prognostic tool for several types of cancer including breast cancer
[5,6]. Thus, the production of antibody against MT2A protein is important for
the study of the molecular basis of metallothionein in carcinogenesis [7].In the present paper we describe the expression of recombinant human
MT2A protein in Escherichia coli. Its expression, purification and
immunization were optimized for the production of MT2A antibody.
Materials and Methods
Animals and cell lines
three-month-old
healthy, parasite- and disease-free New Zealand white rabbits were purchased
from the Centre for Animal Breeding, Nanjing Agricultural University (Nanjing,
China) and used for polyclonal antibody production. Animal studies were
conducted with high standard animal welfare and approved by the Animal Care and
Use Committee, College of Life Sciences, Nanjing University (Nanjing, China).
Human embryonic kidney 293 (HEK293) cells were obtained from the
Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological
Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in
Dulbeccos modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum at 37 ?C in 5% CO2.
Plasmid construction and cell transfection
Plasmid pTWRG–MT2A was used to express the recombinant GST–MT2A
fusion protein. The human heart cDNA library, which was in pACT2 vector, was
obtained from Clontech (San Jose, USA). Polymerase chain reaction (PCR)
amplification was carried out with plasmid pACT2, which
contained MT2A in vector, using upstream primer 5‘–Ccgggatcctcatatggccatgga-3‘ and
downstream primer 5‘-Cggctcgagtcacattatttcataga-3‘.
The MT2A gene encoding 60 amino acid residues was amplified by PCR, and
inserted into pTWRG between the BamHI and XhoI sites [8]. The MT2A
gene was digested with BamHI and XhoI from pACT2 vector and
inserted into pRK5-Flag. PCR was carried out to screen for positive clones,
which was then confirmed by DNA sequencing (Bocai, Shanghai, China). The
plasmid pRK5-MT3 for the expression of metallothionein 3 was constructed
previously in our laboratory by Dr. Wei-Juan ZHENG [9]. Competent E. coli
BL21(DE3) cells were transformed with recombinant plasmid pTWRG-MT2A
according to the manufacturer’s protocol (Pharmacia Biotech, Uppsala, Sweden).
For western blot experiments,
HEK293 cells were transfected with pRK5-MT2A (3 mg) or pRK5-MT3 (3 mg) in 60 mm
dishes using the calcium phosphate transfection method.
GST-MT2A fusion protein preparation
The transformed E. coli cells were grown overnight in 100 ml
Luria Bertani medium containing 100 mg/ml ampicillin. For the large-scale
preparation of GST-MT2A fusion protein, a 20 ml aliquot of the overnight
culture was added to 1 liter of fresh medium in 2 liter conical flasks and
shaken at 225 rpm at 37 ?C. Three hours later, when the bacterial culture
reached a cell density (A600) of 1, MT2A was induced by
the addition of isopropyl ?-D-thiogalactopyranoside (final
concentration of 0.1 mM) for 4 h. A total of 50 ml of culture was pelleted by
centrifugation and washed with phosphate-buffered saline (PBS) (137 mM NaCl,
2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Cells were then resuspended in PBS
and sonicated with ultrasound (550 Sonic Dismembrator; Glen Mills, Clifton,
USA) (1 min, power 4, 50% duty cycle). Supernatant was collected after 5 min of
centrifugation at 14,000 g and the pellet was used for protein
purification by sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
Anti-MT2A antibody production
Proteins were loaded onto 12% SDS-PAGE for electrophoretic
separation. The gel was stained with Coomassie blue then completely destained
with 5% (v/v) methanol and 7% (V/v) acetic acid. The expected target
protein was visualized and labeled, then a 33 kDa fusion protein band was cut
out from the gel. The gel slice was minced then gently tapped into the top of a
3 ml syringe, to which a micro-emulsifying needle was attached. Keeping the
syringe horizontal, 200 ml of PBS solution was carefully introduced to the barrel of the
syringe, and the plunger was inserted. Next, 200 ml of Freunds adjuvant was
drawn into a 1 ml syringe and transferred into the needle end of a second 3 ml
syringe. The two plungers are pushed alternately to mix the components of the
two syringes. This mixture was injected subcutaneously into the neck region of
the rabbit. Four injections totaling 100 mg of MT2A fusion protein in
Freunds adjuvant were given at days 0, 14, 28 and 56 and the final bleeding
was taken at day 90.
Purification of anti-MT2A immunoglobulin G (IgG)
For affinity separation of IgG, protein A-agarose was washed twice
with IgG-binding buffer to remove sodium azide. Thirty microliters of IgG or
plasma sample was added to 270 ml of washed protein A-agarose. The contents were mixed and incubated
at room temperature for 10 min in a protein A affinity column. The agarose
resin was washed twice with binding buffer (10 mM Tris, pH 7.5) to remove
unbound components completely. The agarose resin was eluted with 300 ml of elution
buffer (0.1 M glycine buffer, pH 2–3), the eluent was collected and immediately
neutralized to physiological pH by adding 1.0 M Tris, pH 7.5 [10]. Antibody
solution was adjusted to 1 mg/ml, which is an ideal concentration both for its
stability and for many practical applications. Purified antibody was stored at –20 ?C with 0.02%
sodium azide.
Western blot analysis
For Western blot experiments, HEK293 cells transfected with pRK5-MT2A
or pRK5-MT3 were solubilized with 0.5 ml of lysis buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate) on ice for 30 min.
Insoluble material was removed by centrifugation at 12,000 g for 10 min
at 4 ?C. The supernatants were collected, their protein concentration was
measured using the Bradford method, and 30 mg of supernatants was used
for western detection. E. coli
lysate expressing GST was used as the control. Protein extracts from HEK293
cells and E. coli were separated by 15% SDS-PAGE then
electrophoretically transferred to nitrocellulose membranes (Hybond C;
Amersham, Uppsala, Sweden). Membranes were blocked with 5% non-fat milk for 1 h
then incubated with anti-MT2A polyclonal antibody (1.0 mg/ml) for 1 h at room
temperature. The membrane was washed three times with PBS Tween-20, followed by
incubation for 1 h with horseradish peroxidase conjugate of goat antirabbit IgG
(0.2 mg/ml; Santa Cruz Biotechnology, Santa Cruz, USA). The membrane was
washed then developed with enhanced chemiluminescence reagent (Amersham Life
Science) and exposed to Kodak X-Omat Blue film (NEN Life Science, Boston, USA).
Immunofluorescence assay
HEK293 cells were grown in DMEM (HyClone, Gaithersburg, USA) supplemented
with 10% fetal bovine serum (Hyclone), penicillin, and streptomycin. Cells were
transiently transfectd with pRK5-MT2A by the standard calcium phosphate method
24 h after mounting on a glass cover slip. Fort-eight hours after transfection,
cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton
X-100 for 5 min, and blocked with 3% bovine serum albumin in PBS for 1 h. After
incubation for 1 h with 1/100 diluted anti-MT2A rabbit polyclonal antibody,
cells were then incubated with Cy3-conjugated anti-rabbit IgG antibody
(Sigma-Aldrich, St. Louis, USA) for 1 h. As a control, cells incubated with 3%
bovine serum albumin in PBS, without anti-MT2A rabbit polyclonal antibody and
detected from Cy3-conjugated antirabbit IgG antibody (Sigma-Aldrich). Nuclei
were counterstained with 4‘,6-diamidino-2-phenylindole. Images were
acquired and processed using AxioVision 3.1 software and an Axioplan 2 imaging
microscope (Carl Zeiss, Oberkochen, Germany).
Results and Discussion
In this study, we describe the expression and purification of a
recombinant MT2A protein as well as the production and characterization of the
antiserum directed against a human MT2A protein.We amplified the MT2A encoding region by PCR and cloned the PCR
product in pTWRG plasmid (Fig. 1) and transformed E. coli cells with
the recombinant plasmid. SDS-PAGE analysis showed that there was an obvious
additional band with a molecular weight of approximately 33 kDa, which was
consistent with the expected molecular weight of GST-MT2A fusion protein,
compared with the bacteria-containing empty vector, and the expression level
was approximately 30% of total cellular proteins (Fig. 2). GST-MT2A
protein, however, was highly insoluble and this insolubility has already been
noticed for other metallothionein proteins expressed in E. coli [9,11],
so GST affinity chromatography could not be used for purification. Therefore,
we separated this insoluble protein by SDS-PAGE, and cut the recombinant
protein band from the gel and used the protein/gel mixture to immunize rabbits.We generated the anti-MT2A polyclonal serum by repeating the
immunization of rabbits with the protein/gel method [12]. After final bleeding,
serum was purified by protein A-affinity resin. A total of 90 mg of antibody
was obtained from a rabbit with 85% purity, which was analyzed by SDS-PAGE (Fig.
3). As shown in Fig. 3, there were two main protein bands observed
in the gel, one was approximately 27 kDa and the other 56 kDa. This might
represent the heavy and light chains of antibody IgG.Western blot analysis carried out with the polyclonal antiserum
revealed a 7 kDa band, which corresponds to the MT2A (Fig. 4). When
GST-MT2A expressed in E. coli was subjected to western blot analysis, we observed three bands whose
molecular weights were 33 kDa, 26 kDa and 7 kDa. The 33 kDa band corresponds to
GST-MT2A, whereas the 26 kDa and 7 kDa bands correspond to the degraded GST and
MT2A proteins released by bacterial protease, respectively (Fig. 5). As
the polyclonal antiserum was generated with GST-MT2A fusion protein, the
resulting anti-MT2A antibody also recognized GST protein. It could react
recombinant GST protein (Fig. 5). MT3 expressed in 293T cells was
harvested and analyzed by western
blot with anti-MT2A antibody. We did not observe specific binding for MT3, even
though its molecular weight was approximately 7 kDa [9]. This reveals that
obtained anti-MT2A antibody can detect MT2A in western blotting and it can distinguish MT2A from MT3. The
polyclonal antisera generated could also efficiently detect MT2A cellular
distribution during immunofluorescent microscopic analysis in MT2A-transfected
cells (Fig. 6).In this article we described a simple and low-cost method for
producing polyclonal antibody. The manufacture of IgG preparations by the
method used in our laboratories is effective and reliable, as proven by western blot analysis and
immunofluorescence assay. The production of anti-MT2A antibody provides a
useful tool for further in-depth investigation of the biological function and
distribution of MT2A in the development of various tissues under specific
physiological or pathological conditions.
References
1 Liang Z, Yang Z. Identification
and characterization of a novel gene EOLA1 stimulating ECV304 cell
proliferation. Biochem Biophys Res Commun 2004, 325: 798802
2 Palmiter RD. The elusive
function of metallothioneins. Proc Natl Acad Sci USA 1998, 95: 8428–8430
3 Hamer DH. Metallothionein.
Annu Rev Biochem 1986, 55: 913–951
4 Woo ES, Monks A, Watkins
SC, Wang AS, Lazo JS. Diversity of metallothionein content and subcellular
localization in the National Cancer Institute tumor panel. Cancer Chemother
Pharmacol 1997, 41: 61–68
5 Douglas-Jones AG, Schmid
KW, Bier B, Horgan K, Lyons K, Dallimore ND, Moneypenny IJ et al.
Metallothionein expression in duct carcinoma in situ of the breast. Hum
Pathol 1995, 26: 217–222
6 Goulding H, Jasani B,
Pereira H, Reid A, Galea M, Bell JA, Elston CW et al. Metallothionein
expression in human breast cancer. Br J Cancer 1995, 72: 968–972
7 Zhang L, Zhou W,
Velculescu VE, Kern SE, Hruban RH, Hamilton SR, Vogelstein B et al. Gene
expression profiles in normal and cancer cells. Science 1997, 276: 1268–1272
8 Hua ZC, Zhang HX. A novel
method for enhancing solubility of recombinant protein products in E. coli.
China Patent ZL01127171.X
9 Zheng WJ, Wu F, Zhuang
HQ, Lu C, Yang F, Ma WL, Hua ZC. Expression of human metallothionein III and
its metalloabsorption capability in Escherichia coli. Prep Biochem
Biotechnol 2004, 34: 265–278
10 Walker JM. The Protein
Protocols Handbook. 2nd ed. Totowa: Humana Press Inc. 2000
11 Yamazaki S, Nakanishi M,
Hamamoto T, Hirata H, Ebihara A, Tokue A, Kagawa Y. Expression of human
metallothionein-II fusion protein in Escherichia coli. Biochem Int 1992,
28: 451–460
12 Carstens BP, Perry ME. Purification of a
fragment of MDM2 for production of polyclonal antisera. Protein Expr Purif
2001, 21: 361–365