Cellular & Molecular Immunology 471

Article Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

Faiz MMT Marikar1, Dingyuan Ma1, Jianqiang Ye1, Bo Tang1, Weijuan Zheng1, Jing Zhang1, Min Lu1 and Zichun Hua1, 2

The wild-type human Fas-associated (FADD) protein was expressed as a His-tag fusion protein in Escherichia coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refolded and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD protein was allowed the production of high titre polyclonal antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays. Cellular & Molecular Immunology. 2008;5(6):471-474.

Key Words: FADD, His-tag fusion protein, polyclonal antibody, immunofluorescence assay

Introduction and was a prognostic factor for poor response to chemotherapy (9, 10). The above findings suggest that FADD Fas is a member of the (TNFR) detection tool of FADD protein is indispensable for the family. It consists of an extra cellular domain with extensive purpose and for further investigating the biological and clinic significance and mechanism of human FADD cysteine-rich repeats and a cytoplasmic tail containing a protein. death domain (1, 2). Fas clustering recruits the Fas-associated The present paper described the expression of death domain (FADD) adapter protein and forms the recombinant human FADD protein in Escherichia coli. death-inducing signalling complex (DISC), causing the Recombinant FADD was expressed as a polyhistidine fusion activation of -8 (3). FADD is known mainly for its protein and purified by one step Ni2+ affinity chromato- death receptor adaptor function at the (4-6). graphy under denatured condition. After optimization of its FADD has also been shown to regulate cell proliferation in expression and purification, polyclonal antibody against addition to its function in (3). human FADD was produced, its application in immuno- The human FADD protein contains 208 amino acids, and assays was carefully characterized. All the studies indicated its molecular weight is approximately 27 kDa. FADD has the that FADD antibody produced in the present study is trend of aggregation and thus tends to form insoluble applicable to various immunodetection of human FADD inclusion bodies during its expression in Escherichia coli (7). protein. Recently, the structure of hFADD (residues 1-191) in solution was well revealed by NMR. A point mutation Phe 25 → Tyr was introduced to suppress the self-aggregation of FADD. Materials and Methods Recently increasing evidence showed that absent or low FADD expression was found in different types of tumor cells Animals and cell lines Three-month-old healthy, parasite- and disease-free New Zealand white rabbits used for polyclonal antibody 1The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu production were purchased from the Centre for Animal Center of Hepatobiliary Diseases, Nanjing University, Nanjing 210093, Breeding, Nanjing Agricultural University. All experimental China; protocols were in accordance with the requirement of the 2Corresponding to: Dr. Zichun Hua, College of Life Sciences, Nanjing Animal Care and Ethics Committee, College of Life University, 22 Hankou Road, Nanjing 210093, China. Tel & Fax: Sciences, Nanjing University, and animal studies were +86-25-8332-4605, E-mail: [email protected] conducted with high standard. Human embryonic kidney 293 Received Aug 18, 2008. Accepted Dec 2, 2008. cell (HEK 293) was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Copyright © 2008 by The Chinese Society of Immunology Sciences and cultured in DMEM medium supplemented with

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10% fetal calf serum at 37°C in 5% CO2. filtration using Amicon 8200 stirred cell (Amicon, Millipore, Bedford, MA, USA) with Amicon YM10 membrane (cut off Recombinant plasmid construction and cell transfections = 10,000). At least three dilution/concentration steps were The pCDNA 3.1-hFADD plasmid containing human FADD performed at 4C. The final product was stored in multiple coding sequence was previously constructed in our lab by Dr. aliquots and frozen at -80C. Protein concentration was Jian-Qiang Ye (11). The plasmid was digested with BamH I determined by Bradford method. The total bacterial protein and Xho I to release human FADD gene. Unidirectionally and purified human FADD were analyzed by 12% SDS- cloning at the BamH I and Xho I site was then performed in a PAGE. pET28a plasmid (Novagen, Madison, WI, USA), without the changes of the open reading frame of FADD gene. PCR was Anti-FADD antibody production performed to screen for positive clones, and then a positive Prior to a course of immunizations, a 2-3 ml of test bleed clone was confirmed by DNA sequencing (Bocai Company, was taken from the rabbit to provide a source of preimmune Shanghai, China). Competent Escherichia coli BL21 (DE3) antiserum. The human FADD protein containing 500 g was cells were transformed with recombinant plasmid. For mixed with the Freund’s adjuvant according to the Western blot experiments, HEK 293 cells were transfected manufacturer’s instructions to achieve a final volume of 0.5 with pcDNA 3.1-hFADD in 60-mm dishes using calcium ml/injection. Keeping the syringe horizontal, 300 l of PBS phosphate transfection method. solution which contained the human FADD was carefully

introduced to the barrel of the syringe, and the plunger was Expression of human FADD fusion proteins His-tagged human FADD was expressed as inclusion bodies inserted. Next, 200 l of Freund’s adjuvant was drawn into a in E. coli BL21 (DE3) cells (Novagen, Madison, WI, USA). 1-ml syringe and transferred into the needle end of a second A single colony transformed with pET28a His-FADD 3-ml syringe. The two plungers were pushed alternatively to plasmid was grown overnight at 37C in 20 ml of LB mix the components of the two syringes. This mixture was injected subcutaneously into the neck region of the rabbit. A medium containing 50 g/ml kanamycin. The culture was total of four injections of renatured FADD protein in then inoculated into 1 L of fresh liquid LB medium Freund’s adjuvant were performed at days 0, 14, 28 and 56 containing 50 g/ml kanamycin and once the cells reached before final bleeding was taken at day 90. an A of 0.9-1.0, expression of human FADD was induced 600 at 37C for an additional 4 h by adding IPTG to a final Purification of anti-FADD IgG concentration of 0.6 mM. The cells were pelleted by Before affinity separation of IgG, protein A-agarose was centrifugation at 6,000 rpm at 4C for 10 min. washed twice with IgG binding buffer to remove sodium

Purification of human FADD fusion proteins by a denatured azide. To 270 l of washed protein A-agarose beads, 30 l of method IgG or plasma samples were added. The contents were mixed The bacterial pellet was re-suspended in a volume of 40 ml and incubated at room temperature (RT) for 10 min for buffer A (50 mM sodium phosphate buffer, pH 8.0, 150 mM antibody binding. The agarose resin was washed twice with NaCl, 1 mM PMSF) and ultrasonicated for 15 min on ice. binding buffer (10 mM Tris, pH 7.5) to remove unbound The insoluble fraction was washed with buffer B (50 mM components. The agarose resin was eluted with 300 l of sodium phosphate buffer, pH 8.0, 150 mM NaCl, 1% Triton elution buffer (0.1 M glycine buffer, pH 2-3); the eluent was X-100) by stirring with the magnetic agitator for 2 h. After collected and immediately neutralized to physiological pH centrifugation for 20 min at 12,000 rpm and 4C, the pellet by adding 1.0 M Tris, pH 7.5. Antibody solution was of inclusion bodies was re-suspended in 40 ml buffer C (50 adjusted to 1 mg/ml, which is an ideal concentration for its mM sodium phosphate, pH 8.0, 150 mM NaCl, 8 M urea) stability and for many practical applications. Purified and dissolved by ultrasonic liquid processor (Sonic antibody was stored at -20°C with 0.02% sodium azide (12). Dismembrator 550, Fisher Scientific, USA) for 15 min, followed by centrifugation ( 12,000 rpm, 20 min, 4C). The Western blot analysis supernatant was added to the Ni2+ affinity column (1.6 cm × For Western blot experiments, HEK 293 cells transfected 5 cm) (Sigma, St, Louis, USA) equilibrated with buffer C. with pcDNA-hFADD plasmid were lysed with 0.5 ml of lysis The Ni2+ column was washed with buffer D (50 mM sodium buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% phosphate, pH 8.0, 20 mM imidazole, 150 mM NaCl) to Nonidet P-40, and 0.5% sodium deoxycholate) on ice for 30 remove nonspecific binding proteins. The His6-tagged fusion min. Insoluble material was removed by centrifugation for protein (human FADD) was eluted with buffer E (50 mM 10 min at 12,000 g at 4°C. The supernatants were collected, sodium phosphate, pH 8.0, 500 mM imidazole, 150 mM their protein concentrations were measured using the NaCl). Refolded human FADD was produced by drop-wise Bradford method and 30 g lysates were used for Western adding the denatured human FADD into 0.5 L of refolding blot detection. Protein extracts from HEK 293 cells were buffer (20 mM Tris-HCl, pH 8.0, 10% sucrose, 1 mM EDTA) separated by 15% SDS-PAGE and then electrophoretically over the period of 3-4 h at room temperature. The final urea transferred to nitrocellulose membranes (Hybond C, concentration in the refolding buffer was 0.16 M. The Amersham, USA). Membranes were blocked with 5% remaining urea and imidazole were removed by ultra- non-fat milk for 1 h and then incubated with anti-FADD (1.0

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A B 2-phenylindole (DAPI). Images were acquired and processed 12 M kDaM kDa 1 using AxioVision 3.1 software on Carl Zeiss Axioplan 2. 97 97 66 Results and Discussion 66 43 31 In this study, we described the expression and purification of 43 a recombinant human FADD protein as well as the production and characterization of the antiserum directed from the refolded human FADD protein. 31 14 The entire open reading frame of the human FADD from pcDNA 3.1-hFADD was sub-cloned into pET28a plasmid CD (Novagen, Madison, WI, USA). E. coli BL21 (DE3) strain 12 12 3 transformed with this recombinant vector allowed the 28 kDa expression of a large amount of His-FADD fusion protein. FADD FADD 28 kDa His-FADD protein, however, was highly insoluble and the GADHP 36 kDa majority was found in the post-sonication pellets. This insolubility has already been noticed for other FADD Figure 1. Expression and purification of the recombinant proteins expressed in E. coli (13). The addition of 8 M urea human FADD. (A) SDS-PAGE analysis of recombinant hFADD prior to sonication solubilized most of the protein. Refolded protein. Lane M, molecular weight marker; Lane 1 represents the human FADD was produced by drop-wisely adding the total bacterial protein from IPTG-induced BL21 (DE3) containing the denatured human FADD into refolding buffer, and the final recombinant expression vector pET28a His-FADD; Lane 2, the urea concentration in the refolding buffer was 0.16 M. The purified human FADD. (B) Purification of anti-FADD antibody. yield of purified protein was estimated at 23 mg/L of culture Lane M, molecular weight marker; Lane 1, IgG elution. (C) Western for the denatured His-FADD fusion protein. The use of 8 M blot analysis (anti-FADD) of recombinant proteins expressed in E. urea markedly increased the amount of eluted protein (Figure coli. Lane 1, His-FADD expressed in E. coli; Lane 2, pET28a 1A). Extensive dialysis of the protein against refolding buffer control induced by IPTG in E. coli. (D) Thirty g proteins were was then necessary to remove the excess urea for proper separated on a 10% SDS-PAGE and Western blot analysis was done against anti-FADD (28 kDa) and GAPDH (36 kDa) was included as refolding of the human FADD protein. This step, however, a control. Lane 1, lysates of FADD deficient ; Lane 2, resulted in a slow protein precipitation. lysates of Jurkat cells; Lane 3 lysates of 293 T cells. After SDS-PAGE analysis, there was an obviously homogenous band corresponding to molecular weight (MW) of around 31 kDa, which is consistent with the expected MW of His-FADD fusion protein (Figure 1A), as compared with g/ml) polyclonal antibody for 1 h at room temperature. the bacteria containing empty vector (data not shown). Then membrane was washed three times with phosphate Finally, denatured FADD was refolded by the dilution buffered saline Tween-20 (PBST) and followed by method and used for immunizing rabbits. incubation for 1 h with HRP conjugate of goat anti-rabbit Anti-FADD polyclonal serum was generated by repeated IgG (0.2 g/ml; Santa Cruz Biotechnology Inc, USA). The immunization of rabbits with full-length human FADD membrane was washed and then developed with enhanced protein (14). Titration of antiserum was performed by ELISA chemiluminescence reagent (Amersham Life Science Inc., assay with purified FADD-protein coated onto microplates. A USA) by exposure to Kodak X-Omat Blue film (NEN life high titre of anti-FADD antibodies was detected after the science Products, USA). third injection. After final bleeding, serum was purified by protein A affinity resin; a total of 80 mg antibody was Immunofluorescence assay obtained from one rabbit with 90% purity, which was HEK 293 cells were grown in DMEM (HyClone, USA) analyzed by SDS-PAGE (Figure 1B). In Figure 1B, there are supplemented with 10% FBS (Hyclone, USA), penicillin, two main protein bands observed in the gel, corresponding to and streptomycin. Cells were transiently transfected with the molecular weight of 56 kDa and 27 kDa respectively. The pcDNA-hFADD by the standard calcium phosphate method bands represent the heavy and light chain of IgG molecules. at 24 h after mounting on glass cover slip. At 48 h post- In order to confirm the specificity of the purified transfection, cells were fixed with 4% paraformaldehyde and antibodies, Western blot analysis carried out with the permeabilized with 0.2% Triton X-100 for 5 min, and polyclonal antibody revealed a 28-kDa band which blocked with 3% BSA in PBS for 1 h. After 1-h incubation corresponds to FADD protein (Figures 1C and 1D). with 1/100 diluted rabbit anti-FADD polyclonal antibody, According to Figure 1D, endogenous FADD expression can cells were then incubated with Cy3-conjugated anti-rabbit be detected by anti-FADD polyclonal antibody. It will be IgG (Sigma, USA) for 1 h. As a control, cells incubated with specifically detected FADD due to FADD-deficient Jurkat 3% BSA in PBS, without rabbit anti-FADD polyclonal cells did not exhibit the band. FADD antibodies, which are antibody. Nuclei were counterstained with 4, 6-diamidino produced in our lab, also recognized FADD in other cell lines,

Volume 5 Number 6 December 2008 474 FADD Expression and Its Antibody Preparation

Cy3 DAPI Merged

Anti-FADD (Rabbit)

Control

Figure 2. Immunoflurorescence microscopy of FADD in FADD-transfected HEK293 cells. HEK293 cells transfected with pcDNA-hFADD were stained with rabbit anti-FADD antibody and detected with Cy3-conjugated rabbit antibody. Row A incubated with rabbit anti-FADD antibody, and row B incubated with 3% PBS in BSA and without anti-FADD antibody.

including non T cells, primary cells from patients and Wallach D. A novel protein that interacts with the death domain epithelial cells (data not shown). The polyclonal antiserum of Fas/APO1 contains a sequence motif related to the death could also efficiently detect FADD’s cellular distribution domain. J Biol Chem. 1995;270:7795-7798. during immunoflurorescent microscopic analysis in FADD- 5. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death transfected cells (Figure 2). Cellular distribution of FADD domain of Fas and initiates apoptosis. Cell. 1995;81:505-512. further detected from using Jurkat and FADD deficient 6. Kischkel FC, Hellbardt S, Behrmann I, et al. Cytotoxicity- Jurkat cells in this experiment (data not shown). It reveals dependent APO-1 (Fas/CD95) - associated proteins form a that the low-cost FADD antibody can be used to detect death-inducing signaling complex (DISC) with the receptor. FADD protein in primary cells. EMBO J. 1995;14:5579-5588. In this article, it was described the productions of both 7. Perez D, White E. E1B 19K inhibits Fas-mediated apoptosis recombinant human FADD and polyclonal antibody against through FADD-dependent sequestration of FLICE. J Cell Biol. human FADD with high specificity via a simple and 1998;141:1255-1266. cost-effective method. It will be of great application potential 8. Mishima K, Nariari Y, Yoshimura Y. Carboplatin induces Fas for various immunodetection of human FADD protein and (APO-1/CD95) dependent apoptosis of human tongue for the investigation of FADD’s function in physiological carcinoma cells; sensitization for apoptosis by upregulation of FADD expression. Int J Cancer. 2003;105:593-600. and pathological conditions. 9. Tourneur L, Delluc S, Levy V, et al. Absence or low expression of fas-associated protein with death domain in acute myeloid Acknowledgements leukemia cells predicts resistance to chemotherapy and poor outcome. Cancer Res. 2004;64:8101-8108. 10. Tourneur L, Mistou S, Michiels FM, et al. Loss of FADD The project was financially supported by the following funds protein results in a biased Fas-signaling pathway and correlates to Zi-Chun Hua: the National Nature Science Foundation of with the development of tumoral status in thyroid follicular China (No. 30425009, 30600320, 30730030, 30330530, cells. Oncogene. 2003;22:2795-2804. 30270291), Jiangsu Provincial Nature Sciences Foundation 11. Ye J, Shao H, Ma D, et al. Development of a monoclonal (No. BK2007715) and Jiangsu Provincial Department of antibody-based sandwich ELISA for detection of human FADD. Health (No. H200524, H200742). Ying Yong Sheng Wu Hua Xue Yu Sheng Wu Ji Shu. 2008;50:143-146. 12. Suzuki A, Araki T, Miura M, Tsutomi Y. Functional absence of References FADD in PLC/PRF/5 hepatoma cells: possible involvement in the transformation to helatoma in HBV-infected hepatocytes. 1. Nagata S. Apoptosis by death factor. Cell. 1997;88:355-365. Pro Soc Exp Biol Med. 1999;221:72-79. 2. Nagata S, Golstein P. The Fas death factor. Science. 1995; 13. Gajate C, Molinedo F. Cytoskeleton-mediated death and 267:1449-1456. concentration in lipid rafts forms apoptosis-promoting clusters 3. Osborn SL, Sohn SJ, Winoto A. Constitutive phosphorylation in cancer chemotheraphy. J Biol Chem. 2005;280:11641-11647. mutation in Fas-associated death domain (FADD) results in 14. Carstens BP, Perry ME. Purification of a fragment of MDM2 for early defects. J Biol Chem. 2007;282:22786-22792. production of polyclonal antisera. Prot Expr Puri. 2001;21: 4. Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, 361-365.

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