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 death domain (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 tumor necrosis factor receptor (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 caspase-8 (3). FADD is known mainly for its protein and purified by one step Ni2+ affinity chromato- death receptor adaptor function at the cell membrane (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 apoptosis (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 Volume 5 Number 6 December 2008 472 FADD Expression and Its Antibody Preparation 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 4C. The final product was stored in multiple coding sequence was previously constructed in our lab by Dr. aliquots and frozen at -80C. 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 37C 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 37C 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 4C 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 4C, 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, 4C). 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.