Research Papers. TNF-α. L929 LPS Carswell [1] BCG. pet-41d TNF).TNF. NEB TNF-α cdna Invitrogen DNA. 75 ku TNF TransTaq High %

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1 Research Papers Progress in Biochemistry and Biophysics 2009, 36(4): 424~430 wwwpibbaccn * TNF-α 1, 2) 1) 1)** ( 1) ) ) TNF-α TNF-α TNF-α TNF-α TNF-α TNF-α 9C6 TNF-α 29~40 TNF-α 9C6 TNF-α L929 9C6 TNF-α TNF-α Q51 R392 DOI: /SPJ Carswell [1] BCG L929 LPS pet-41d pet-41a GST pyds (tumor necrosis factor, pyd1 (Invitrogen ) [2] TNF)TNF pmd19-t TaKaRa T4 DNA [3] TNF-α 55 ku TNF 75 ku TNF DNA DNA [4~7] TransTaq High TNF-α Fidelity DNA TNF-α DNA Tiangen Ni GE TNF-α 17 ku Healthcare MTT Sigma 156 TNF-α D MERCK DMEM Anti-V5-FITC TNF-α 79% Invitrogen goat-anti-mouse-igg-fitc TNF-α SouthernBiotech goat-anti-mouse-igg-hrp TNF-α KPL Biotin-anti-mouse-TNF-α TNF-α Streptavidin-HRP Ebioscience TNF-α 12 TNF-α 9C6 TNF-α N C TNF-α TNF-α TNF-α 1 11 DH5α BL21(DE3) EBY100 NEB TNF-α cdna Invitrogen * (863) (2006AA , 2006AA ) ** Tel: , jtang@sun5ibpaccn

2 2009; 36 (4) TNF-α TNF-α 17~28 FACS (1%BSA-PBS) r/min 23~34 29~40 17~ 5 min anti-v5-fitc 40 9C min 1 ml FACS μl FACS DNA FACS calibur V5 (Mutant A) TNF-α 30 (Ser) Ni 12% SDS-PAGE 34 (Leu) TNF-α 17 TNF-α ELISA 34 TNF-α Mutant A 2 mg/l 2 (Asn) 10 PCR (005 mol/l ph 96) TNF-α h, PBS-T(01% Tween 20) (Asn) 37 (Ala) (Mutant B) 31 (Gln) (Asn) 39 (Ala) 2 9C nm A SD 2 ml SD ( 2% ) 30 A 2 1 ml r/min 5 min PBS-T 3 2% BSA-PBS 1 ml SG(selective growth galactose medium) 200 μl 37 1 h PBS-T 3 9C6 ( 2% ) r/min 5 min 10 mg/l 3 2 ml SG h 15 SG ml TNF-α 9C6 200 nmol/l 4 30 min 1 ml FACS goat anti-mouse-igg-fitc 4 30 min 1 ml FACS μl FACS 16 TNF-α TNF cdna TNF-α Mutant A DNA BamH Hind pmd19-t BamH Hind pet-41d BL21(DE3) LB Kana + 37 A 06 ~08 01 mmol/l IPTG (isopropyl β-d-1-thiogalactopyranoside)16 16 h (500 mmol/l NaCl, 50 mmol/l Na 2 HPO 4, 20 mmol/l ph 80) 3 2%BSA-PBS 37 1 h PBS-T 3 13 [8] Biotin-anti-mouse- SD(selective TNF-α 100 μl 37 1 h PBS-T 3 growth dextrose medium) Streptavidin-HRP h PBS-T 3 OPD TNF-α 2 mg/l 100 μl 37 1 h, 100 μl 100 PBS 37 1 h PBS-T 3 goat-anti-mouse-igg-hrp

3 426 Prog Biochem Biophys 2009; 36 (4) 37 1 h PBS-T 3 OPD 5 min 2 mol/l 490 nm 2 A 18 9C6 TNF-α L % CO 2 DMEM 10% L /ml D 1 mg/l μl TNF-α C R C6 10 μg/l 9C6 20 mg/l 2 TNF-α min 9C6 TNF-α 10 μl μl 17~40 ( 1) TNF-α 3 TNF-α 9C6 37, 5%CO 2 20 h 34 29~40 20 μl MTT (5 g/l) 3 h 150 μl (DMSO), 570 nm A TNF-α 29~40 19 TNF-α LSQRANALLANG ( 2) L929 29~40 30 L /ml (Ser) 34 D 1 mg/l μl 5 μl TNF-α TNF-α Mutant A 100 μl TNF-α TNF-α Mutant A 1 mg/l 3 39 (Asn) TNF-α TNF-α Mutant A 3 37, 5% CO 2 20 h 20 μl MTT(5 g/l) C6 TNF-α TNF-α 9C6 N F1-20 9C6 ( ) 9C6 9C6 17~ ~28 23~ 9C6 29~ 40 9C6 9C6 (Asn) 37 (Leu) (Ala) Mutant A 31 (Gln) 34 (Asn) (Ala) Mutant B TNF-α anti-v5- FITC 5% CO 2 3 h 150 μl 9C6 Mutant A 9C6 (DMSO) 570 nm Mutant B 9C6 ( 3) 9C6 A TNF-α 29~40 Fig 2 FACS histogram of mouse TNF-α fragments displayed on yeast surface, labeled with anti-v5-fitc and 9C6 respectively

4 2009; 36 (4) TNF-α 427 Fig 3 Flow cytometric analysis of mouse TNF-α(WT) and mouse TNF-α mutants(mutant A and Mutant B) displayed on yeast cell surface labeled with anti-v5-fitc and 9C6 respectively WT: wild-type 22 TNF-α TNF-α Mutant A TNF-α cdna PCR TNF-α pmd19-t BamH Hind T4 DNA pet-41d pet-41d-m- TNF pet-41d-m-tnf-mutant A BL21(DE3) IPTG TNF-α Ni 18 ku TNF-α Mutant A ( 4) 23 ELISA TNF-α TNF-α C6 goat-anti-mouse-igg-hrp (1 5000) OPD 490nm A 9C6 ( 5a)

5 428 Prog Biochem Biophys 2009; 36 (4) Biotin-anti-mouse-TNF-α Streptavidin-HRP TNF-α Mutant A 9C6 TNF-α 10 μg/l TNF-α TNF-α ( 5b) TNF-α L mg/l 9C6 ( 6a) L929 L929 9C6 24 9C6 TNF-α 37 5% CO 2 20 h L μl MTT(5 g/l) 37 5% CO 2 3 h DMSO 570 nm 10 μg/l TNF-α L929 A TNF-α Mutant A 0~20 mg/l 9C6 L h 20 μl MTT(5 g/l), 37, 5% CO 2 (DMSO) 570 nm A TNF-α Mutant A TNF-α 9C6 TNF-α 3 h ( 6b) TNF-α Ala) 9C6 ( 3) TNF-α Mutant A TNF-α ELISA 9C6 TNF-α 9C6 ( 5a) TNF-α TNF-α L929 [9, 10] [11, 12] Mutant A L929 ( 9C6 6b) 3 TNF-α TNF-α ( 6a) Western-blot 9C6 TNF-α( ) 9C6 TNF-α (Ser30/Asn34/Leu37 Ala) 9C6 Mutant B(Gln31/Asn34/Asn39 TNF-α TNF-α 79% ( 7) TNF-α L929 TNF-α 29~ [13] 36 84~91 143~149 9C6 TNF-α 29~40 TNF-α ( 2) TNF-α TNF-α TNF-α [14] 29~40 Mutant A TNF-α

6 2009; 36 (4) TNF-α 429 TNF-α TNF-β TNF TNF-α 7~11 17~23 30~39 42~ [9] ~ ~142-9C6 TNF-α 9C6 TNF-α ( ) TNF-β TNF-α 29~40 TNF-α ( 7) TNF-α TNF-β Human TNF-α Mouse TNF-α Human TNF-β 29~40aa Human TNF recaptor Fig 7 Comparison of mouse TNF-α with human TNF-α and the complex of human TNF-β and TNF receptor The three-dimensional structures of human and mouse TNF-α are shown and the 9C6 binding site on mouse TNF-α is indicated by an arrowthe three-dimensional structures of TNF-α and TNF-β are strikingly similar and they bind to the same receptorsthe 9C6 binding site of mouse TNF-α corresponds to the TNF receptor binding site on human TNF-β TNF-α TNF-α TNF-α [15] TNF-α (33): 19509~19519 TNF-α TNF-α TNF-α 1 Carswell E A, Old L J, Kassel R L, et al An endotoxin-induced serum factor that causes necrosis of tumours Proc Natl Acad Sci USA, 1975, 72(9): 3666~ Old L J Tumor necrosis factor Sci Am, 1988, 258(5): 59~75 3 Clark I A How TNF was recognized as a key mechanism of disease Cytokine & Growth Factor Reviews, 2007, 18(3~4): 335~343 4 Beutler B, Cerami A The biology of cachectin/tnf-α primary mediator of the host response Annu Rev Immunol, 1989, 7: 625~ Tracey K J, Wei H, Manogue K R, et al Cachectin/tumour necrosis factor induces cachexia, anemia, and inflammation J Exp Med, 1988, 167(3): 1211~ Loetscher H, Pan Y C, Lahm H W, et al Molecular cloning and expression of the human 55 ku tumour necrosis factor receptor Cell, 1990, 61(2): 351~359 7 Schall T J, Lewis M, Koller K J, et al Molecular cloning and expression of a receptor for human tumour necrosis factor Cell, 1990, 61(2): 361~370 8 Gietz R D, Woods R A Transformation of yeast by lithium acetate/ single-stranded carrier DNA/polyethylene glycol method Methods Enzymol, 2002, 350: 87~96 9 Yone K, Bajard S, Tsunekawa N, et al Epitopic regions for antibodies against tumor necrosis factor α J Biol Chem, 1995, Nagahira K, Fukuda Y, Terakawa M, et al Epitope mapping of monoclonal antibodies to tumor necrosis factor-α by synthetic peptide approach Immunology Letters, 1995, 46(1~2): 135~ Shingarova L N, Sagaidak L N, Berkova N, et al Determination of binding site of anti-tumor necrosis factor-α monoclonal antibody using hybrid and mutant proteins FEBS Letters, 1996, 386(1): 72~ Zhu C S, Liu X S, Feng J N, et al Characterization of the neutralizing activity of three anti-human TNF monoclonal antibodies and prediction of their TNF epitopes by molecular modeling and mutant protein approach Immunology Letters, 2006, 102(2): 177~ Van Ostade X, Tavernier J, Fiers W Structure-activity studies of human tumor necrosis factors Protein Eng, 1994, 7(1): 5~22 14 Tracey D, Klareskog L, Sasso E H, et al Tumor necrosis factor antagonist mechanisms of action: A comprehensive review Pharmacol Ther, 2008, 117(2): 244~ Feldmann M, Maini R N Anti-TNF-α therapy of rheumatoid arthritis: What have we learned? Annu Rev Immunol, 2001, 19: 163~196

7 430 Prog Biochem Biophys 2009; 36 (4) The Study of Mouse TNF-α Functional Domain and Its Neutralizing Antibody Binding Site JIA Jun-Ying 1,2), ZHOU Hong-Zhe 1), TANG Jie 1)** ( 1) Institute of Biophysics, The Chinese Academy of Sciences, Beijing , China; 2) Graduate School of The Chinese Academy of Sciences, Beijing , China) Abstract Tumor necrosis factor alpha(tnf-α) is a multi-functional cytokine that plays a significant role in many autoimmune diseases The key biological functional domain of mouse TNF-α was determined by identifying the binding site of mouse TNF-α neutralizing antibody 9C6 Using yeast surface display technology, it was determined that 9C6 can recognize the linear amino acid fragment 29 ~40 of mature mouse TNF-α protein Through mutagenesis experiments of this TNF-α region, the critical amino acids necessary for 9C6 binding were identified Finally, wild-type mouse TNF-α and mutant variants that loses binding ability to 9C6 were expressed in Escherichia coli, purified, and used in a L929 cell killing assay The assay results proved that the key amino acids for 9C6 binding were consistent with mouse TNF-α functional domain Key words mouse TNF-α, neutralizing antibody, binding site, yeast display DOI: /SPJ *This work was supported by a grant from The Hi-Tech Research and Development Program of China(2006AA , 2006AA ) **Corresponding author Tel: , jtang@sun5ibpaccn Received: July 16, 2008 Accepted: September 22, 2008