Research Papers (EGFR) PCR (MIF) EcoR Not Bgl. Pyrobest Taq. (scfv) Progress in Biochemistry and Biophysics 2009, 36(3): 305~310

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1 Research Papers Progress in Biochemistry and Biophysics 29, 36(3): 35~31 wwwpibbaccn * 1, 2) 1) 1)** ( 1) 111 2) 139) (macrophage migration inhibitory factor MIF) 3 1C3 2A12 4E1 MIF 3 MIF Q5 R392 DOI: 13724/SPJ (EGFR) [4] PCR [5, 6] (MIF) 1 11 EBY1 DH5α pyds pyd1 (Invitrogen ) 12 EcoR Not Bgl Pyrobest Taq (scfv) [1, 2] [3] * (863)(26AA2985) ** Tel: jtang@sun5ibpaccn

2 36 Prog Biochem Biophys 29; 36 (3) 17 MIF MIF 115 N 1 MIF MIF 5 1C3 2A12 4E1 MIF C 1 MIF goat anti-mouse-igg-fitc SouthernBiotech anti-v5-fitc Invitrogen EcoR Not Streptavidin-PE BD Pharmingen 13 [7] SD(selective anti-v5-fitc V5 1C3, 2A12, 4E1 growth dextrose medium) SD 2 ml SD MIF Bgl ( 2% ) 3 A 2 21~24 1 ml 5 r/min 5 min 21~24 MIF 1 ml SG(selective growth galactose medium) 4 MIF-Bgl -1( ( 2% ) 5 r/min 5 min 2 48 h 2 ml SG ) MIF-Bgl -3 ( 58 ~78 ) MIF- 15 PCR 4 MIF SG ml FACS (PBS+1%BSA) 1 5 r/min 5 min 1 1 anti-v5-fitc FACS Calibur V5 MIF 1C3 2A12 4E1 EcoR Not 2 nmol/l 4 3 min 1 ml FACS pyds goat- anti-mouse-igg- FITC 4 3 min Bgl 16 MIF 1C3 2A12 4E1 19 MIF MIF-Bgl -1 MIF-Bgl -2 5 nmol/l 1 min MIF-Bgl -3 MIF-Bgl -4 Bgl 1 nmol/l 3 min 1 ml FACS Streptavidin-PE 4 3 min 1 ml (A) 21~24 FACS 3 DNA 63~72 bp 2A12 MIF 5 bp 3 2 nmol/l 1C3 4E1 MIF 2 min 2A12 PCR 2A12 25 nmol/l 2 98 nmol/l 3 min 1 ml FACS Streptavidin-PE 5 PCR pyds 18 MIF Bgl 16~36 ) MIF-Bgl -2( 37~57 Bgl -4( 77~1 ) Bgl 2 MIF Bgl 2 MIF PCR MIF DH5α Bgl anti-v5-fitc V5 4 3 min FACS 3 DH5α 2A12 MIF 1C3 2A12 4E1

3 29; 36 (3) 37 2 ( 1a) 1C3 2A12 4E1 21 MIF 1C3 2A12 4E1 5 nmol/l 4E1 1 min 1 nmol/l 2A12 Streptavidin- SG MIF PE 1 nmol/l 1C3 Streptavidin-PE ( 1b) 2A12 4E1 5 5 nmol/l 1C3 1 min 2A12 1 nmol/l 1C3 1 nmol/l 1C3 Streptavidin-PE 1C3 4E1 Streptavidin-PE ( 1c) 3 MIF 5 nmol/l 2A12 4E1 1 min (a) Geo mean (b) Geo mean (c) Percent of maximal signal c(bio-2a12)/(nmol L -1 ) Fig 1 Competitive binding of mabs to yeast-displayed MIF (a) Competition of mabs between 1C3 and 2A12/4E1: yeast clone displaying wild-type MIF was first labeled with 5nmol/L 1C3 2A12 and 4E1 respectively then 1nmol/L biotinylated 1C3 was added and finally Streptavidin-PE was used to detect biotinylated 1C3Control was the sample labeled with 1nmol/L biotinylated 1C3 1: Bio-1C3; 2: Bio-1C3+2A12; 3: Bio-1C3+4E1; 4: Bio-1C3+1C3 (b) Competition between 2A12 and 4E1: yeast was labeled with 5nmol/L 4E1 and 1C3 respectively next 1nmol/L biotinylated 2A12 was added 1: Bio-2A12; 2: Bio-2A12+1C3; 3: Bio-2A12+4E1; 4: Bio-1C3+1C3 (c) Yeast displayed MIF incubated with 2 nmol/l 1C3 or 4E1 firstly the following added biotinylated 2A12 with varying concentration : Bio-2A12; : Bio-2A12+1C3; : Bio-2A12+4E1 22 MIF PCR MIF F1-1 F1-8 anti-v5-fitc F1-6 V5 F1-4 F1-2 1C3 2A12 4E1 MIF C MIF F1-1 MIF (human-mif) 1C3 2A12 4E1 1C3 2A12 4E1 MIF N MIF Fig 2 MIF fragments displayed on the yeast cell R C3 surface and tested for binding to MIF mabs, 1C3 2A12 4E1 ( 2) as detected by FACS 3 MIF MIF by flow cytometry +: Binding; -: No binding MIF Human-MIF R R R R R C3 2A12 4E Cells were washed and incubated with 1C3, 2A12 and 4E1 respectively, then labeled with goat anti-mouse-igg-fitc and analyzed

4 38 Prog Biochem Biophys 29; 36 (3) 23 MIF 1C3 2A12 4E1 Table 1 Binding of MIF alanine mutants to mabs Binding of mabs Code Mutants 1C3 2A12 4E1 MIF 4 Bgl B1-1A D17A B1-2A E22A Bgl C3 2A12 4E1 1 M48A 1C3 1C3 M48A I97A 2A12 2 2A12 MIF L27A I38A M48A I97A 4E1 ( 3) B1-3A L27A B1-4A G32A B2-1A I38A B2-2A V43A B2-3A M48A B2-4A S53A B3-1A L59A B3-2A S64A B3-3A G69A B3-4A R74A B4-1A S77A B4-2A G82A B4-3A R87A B4-4A P92A B4-5A I97A : Binding of alanine mutant similar to wild-type; ++: Intermediate binding; +: Weak, but detectable binding; -: Complete loss of binding mab 15 1C3 15 2A E1 WT L27A I38A Mutant M48A I97A mab binding Fig 3 Flow cytometry histograms for alanine mutants of MIF that exhibited loss of binding to mabs Yeast was labeled with 1C3, 2A12 and 4E1 respectivelyrepresentative flow cytometry histograms depicting the mean fluorescence signal of MIF antibody labeling of yeast displayed MIFWT: Wild-type

5 29; 36 (3) 39 3 MIF 3 [8] [9] 4 ( 4)Met48 [5] 1C3 2A12 4E1 MIF a 3 Ile97 2A12 4E1 PCR DNA PCR Leu27 Ile38 PCR 4E1 4E1 b Leu27 PCR Ile97 Ile38 PCR 1 c Western-blot ELISA 17 MIF 3 Met48 Fig 4 Single amino acid mutations in MIF that exhibited loss-of-binding to mabs 1C3, 2A12 or 4E1 Ile97 mutants result in 4E1 loss of binding to MIF 1 MIF N C 1C3 2A12 4E1 1 MIF ( 2) 3 MIF MIF 15~ 1 17 ( 1) DNA ( 1) Met48 mutant partially inhibits 1C3 binding to MIFMet48 and Ile97 mutants result in 2A12 loss of binding to MIFLeu27, Ile38, Met48 and 1 Feldhaus M J, Siegel R W Yeast display of antibody fragments: a discovery and characterization platform J Immunological Methods, 24, 29(1~2): 69~8 2 Weaver-Feldhaus J M, Miller K D, Feldhaus M J, et al Directed evolution for the development of conformation-specific affinity reagents using yeast display Protein Engineering, Design & MIF 4 Bgl Bgl Selection, 25, 18( 11): 527~536 3 Richman S A, Healan S J, Weber K S, et al Development of a novel strategy for engineering high-affinity proteins by yeast display Protein Engineering, Design & Selection, 26, 19(6): 255~264

6 31 Prog Biochem Biophys 29; 36 (3) 4 Cochrana J R, Kima Y S, Olsen M J, et al Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments J Immunological Methods, 24, 287(1~ 2): 147~158 5 Chao G, Cochran J R, Wittrup K D Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display J Mol Biol, 24, 342 (2): 539~55 6 Levy R, Forsyth C M, LaPorte S L, et al Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display J Mol Biol, 27, 365(1): 196~ 21 7 Gietz R D, Woods R A Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method Methods Enzymol, 22, 35: 87~96 8 Nagahira K, Fukuda Y, Terakawa M, et al Epitope mapping of monoclonal antibodies to tumor necrosis factor-alpha by synthetic peptide approach Immunology Letters, 1995, 46(1~2): 135~141 9 Shingarova L N, Sagaidak L N, Berkova N, et al Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins FEBS Letters, 1996, 386 (1): 72~74 A New Strategy for Epitope Mapping by Yeast Surface Display System * JIA Jun-Ying 1,2), WANG Yun-Bo 1), TANG Jie 1)** ( 1) Institute of Biophysics, The Chinese Academy of Sciences, Beijing 111, China; 2) Graduate School of The Chinese Academy of Sciences, Beijing 139, China) Abstract As an effective method of studying soluble protein-protein interactions, yeast display system is now widely used for affinity maturation of single-chain antibodies Due to the strong homology recombination machinery of yeast and the high-throughput nature of FACS detection, a rapid scan for interaction between antigen-antibody pairs could be easily achieved Based on this system, a novel and reliable method for determining conformational epitopes was developed Different fragments of macrophage migration inhibitory factor (MIF) and several point mutations of MIF were displayed on yeast cell surface using homologous recombination technology Three MIF monoclonal antibodies, 1C3, 2A12 and 4E1, were screened for their binding affinity to each displayed peptide Utilizing this technology, the key amino acids of MIF that bind to the MIF monoclonal antibodies were easily identified Key words yeast display, MIF, epitope mapping, homologous recombination DOI: 13724/SPJ *This work was supported by a grant from Hi-Tech Research and Development Program of China(26AA2985) **Corresponding author Tel: , jtang@sun5ibpaccn Received: June 2, 28 Accepted: September 22, 28