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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 305-309 |
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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 Dulbecco�s 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'-Cggctcgagtcacattatttc�ataga-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 β-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 Freund�s 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 Freund�s 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 in�solubility 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.
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