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• W2095777724 abstract "In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K d) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20–51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R1), Bombyx mori (Bt-R175), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R1 or Bt-R175 are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R1 or Bt-R175receptors. Finally, we showed that synthetic peptides homologous to Bt-R1 and scFv73 CDR3 and the scFv73 antibody decreased thein vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R1 and Bt-R175 involved in Cry1A toxin interaction. In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K d) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20–51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R1), Bombyx mori (Bt-R175), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R1 or Bt-R175 are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R1 or Bt-R175receptors. Finally, we showed that synthetic peptides homologous to Bt-R1 and scFv73 CDR3 and the scFv73 antibody decreased thein vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R1 and Bt-R175 involved in Cry1A toxin interaction. B. thuringiensis aminopeptidase N brush border membrane vesicles surface plasmon resonance complementary determinant region polyacrylamide gel electrophoresis single-chain variable fragment HEPES-buffered saline with surfactant P-20 Luria broth nutrient broth Synthetic insecticides cause not only environmental problems, but many have lost their efficacy due to resistance development in the pest insects. Bacillus thuringiensis(Bt),1 a biopesticide, is a viable alternative for the control of insect pests in agriculture and disease vectors of importance in public health. Bt use is also compatible with sustainable and environmentally friendly agricultural practices. Bt produces insecticidal proteins (Cry toxins) during sporulation as parasporal crystals. These crystals are predominantly composed of one or more proteins, also called δ-endotoxins. These toxins are highly specific to their target insect; are safe to humans, vertebrates, and plants; and are completely biodegradable. The three-dimensional structures of Cry3A and Cry1Aa toxins have been resolved by x-ray diffraction crystallography (1Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (647) Google Scholar, 2Grochulski P. Masson L. Borisova S. Pusztai-Carey M. Schwartz J.L. Brousseau R. Cygler M. J. Mol. Biol. 1995; 254: 447-464Crossref PubMed Scopus (462) Google Scholar). The two proteins share many similar features and are composed of three domains. Domain I, extending from the N terminus, a seven-helix bundle, is the pore-forming domain. Domain II consists of three anti-parallel β-sheets, and domain III is a β-sandwich of two anti-parallel β-sheets (1Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (647) Google Scholar, 2Grochulski P. Masson L. Borisova S. Pusztai-Carey M. Schwartz J.L. Brousseau R. Cygler M. J. Mol. Biol. 1995; 254: 447-464Crossref PubMed Scopus (462) Google Scholar). Domains II and III are involved in receptor binding, and domain III additionally protects the toxin from further proteolysis (for reviews, see Refs. 3Pietrantonio P.V. Gill S.S. Lehane M.J. Billingsley P.F. Biology of the Insect Midgut. Chapman and Hall, London1996: 345-372Crossref Google Scholar and 4Schnepf H.E. Crickmore N. Van Rie J. Dereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar). The mode of action of Cry toxins is a multistage process. Crystal toxins ingested by susceptible larvae dissolve in the alkaline environment of the larval midgut, thereby releasing soluble proteins. The inactive protoxins are then cleaved at specific sites by midgut proteases, yielding 60–70-kDa protease-resistant active fragments. The active toxin then binds to specific membrane receptors on the apical brush border of the midgut epithelium columnar cells (5Bravo A. Jansens S. Peferoen M. J. Invertebr. Pathol. 1992; 60: 237-246Crossref Scopus (109) Google Scholar, 6Hofmann C. Vanderbruggen H. Höfte H. Van Rie J. Jansens S. Van Mellaert H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7844-7848Crossref PubMed Scopus (344) Google Scholar). Therefore, receptors on the brush border membrane are a key factor in determining the specificity of Cry toxins. This specific binding involves two steps, a reversible followed by an irreversible one (7Dean D.H. Rajamohan F. Lee M.K. Wu S.-J. Chen X.J. Alcántara E. Hussain S.R. Gene (Amst.). 1996; 179: 111-117Crossref PubMed Scopus (64) Google Scholar). After binding, the toxin apparently undergoes a large conformational change leading to its insertion into the cell membrane (1Li J. Carroll J. Ellar D.J. Nature. 1991; 353: 815-821Crossref PubMed Scopus (647) Google Scholar). The Cry toxin molecules then aggregate through toxin-toxin interactions (8Soberón M. Perez R.V. Nuñez-Valdéz M.E. Lorence A. Gómez I. Sánchez J. Bravo A. FEMS Microbiol. Lett. 2000; 191: 221-225Crossref PubMed Scopus (29) Google Scholar), leading to the formation of lytic pores (8Soberón M. Perez R.V. Nuñez-Valdéz M.E. Lorence A. Gómez I. Sánchez J. Bravo A. FEMS Microbiol. Lett. 2000; 191: 221-225Crossref PubMed Scopus (29) Google Scholar, 9Lorence A. Darszon A. Dı́az C. Liévano A. Quintero R. Bravo A. FEBS Lett. 1995; 360: 217-222Crossref PubMed Scopus (100) Google Scholar, 10Schwartz J.L. Garneau L. Masson L. Brousseau R. Rousseaeu E. J. Membr. Biol. 1993; 132: 53-62Crossref PubMed Scopus (110) Google Scholar), which disrupt midgut ion gradients and the transepithelial potential difference. This disruption is accompanied by an inflow of water that leads to cell swelling and eventual lysis, resulting in paralysis of the midgut and subsequent larval death (3Pietrantonio P.V. Gill S.S. Lehane M.J. Billingsley P.F. Biology of the Insect Midgut. Chapman and Hall, London1996: 345-372Crossref Google Scholar, 4Schnepf H.E. Crickmore N. Van Rie J. Dereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar). A number of putative receptor molecules for lepidopteran-specific Cry1A toxins have been identified. In Manduca sexta, Cry1Aa, Cry1Ab, and Cry1Ac proteins bind to a 120-kDa aminopeptidase N (APN) (11Denolf P. Hendrickx K. VanDamme J. Jansens S. Peferoen M. Egheele D. VanRie J. Eur. J. Biochem. 1997; 248: 748-761Crossref PubMed Scopus (89) Google Scholar, 12Garczynski S.F. Adang M.J. Insect Biochem. Mol. Biol. 1995; 25: 409-415Crossref Scopus (71) Google Scholar, 13Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (358) Google Scholar) and to a 210-kDa cadherin-like protein (Bt-R1) (14Belfiore C.J. Vadlamudi R.K. Osman Y.A. Bulla Jr., L.A. Biochem. Biophys. Res. Commun. 1994; 200: 359-364Crossref PubMed Scopus (30) Google Scholar, 15Vadlamudi R.K. Weber E. Ji I. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1995; 270: 5490-5494Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In Bombyx mori, Cry1Aa binds to a 175-kDa cadherin-like protein (Bt-R175) (16Nagamatsu Y. Toda S. Yagamuchi F. Ogo M. Kogure M. Nakamura M. Shibata Y. Katsumoto T. Biosci. Biotechnol. Biochem. 1998; 62: 718-726Crossref PubMed Scopus (84) Google Scholar, 17Nagamatsu Y. Koike T. Sasaki K. Yoshimoto A. Furukawa Y. FEBS Lett. 1999; 460: 385-390Crossref PubMed Scopus (117) Google Scholar) and to a 120-kDa APN (18Yaoi K. Kadotani T. Kuwana H. Shinkawa A. Takahashi T. Iwahana H. Sato R. Eur. J. Biochem. 1997; 246: 652-657Crossref PubMed Scopus (89) Google Scholar). In Heliothis virescens, Cry1Ac binds to two proteins of 120 and 170 kDa, both identified as APN (20Gill S.S. Cowles E.A. Francis V. J. Biol. Chem. 1995; 270: 27277-27282Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21Oltean D.I. Pullikuth A.K. Lee H-K. Gill S. Appl. Environ. Microbiol. 1999; 65: 4760-4766Crossref PubMed Google Scholar). InPlutella xylostella and Lymantria dispar APNs were identified as Cry1Ac receptors (11Denolf P. Hendrickx K. VanDamme J. Jansens S. Peferoen M. Egheele D. VanRie J. Eur. J. Biochem. 1997; 248: 748-761Crossref PubMed Scopus (89) Google Scholar, 22Luo K. Tabashnik B.E. Adang M.J. Appl. Environ. Microbiol. 1997; 63: 1024-1027Crossref PubMed Google Scholar, 23Lee M.K. You T.H. Young B.A. Cotrill J.A. Valaitis A.P. Dean D.H. Appl. Environ. Microbiol. 1996; 62: 2845-2849Crossref PubMed Google Scholar, 24Valaitis A.P. Lee M.K. Rajamohan F. Dean D.H. Insect Biochem. Mol. Biol. 1995; 25: 1143-1151Crossref PubMed Scopus (102) Google Scholar). All of these receptor molecule proteins are glycosylated (12Garczynski S.F. Adang M.J. Insect Biochem. Mol. Biol. 1995; 25: 409-415Crossref Scopus (71) Google Scholar, 15Vadlamudi R.K. Weber E. Ji I. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1995; 270: 5490-5494Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Nagamatsu Y. Toda S. Yagamuchi F. Ogo M. Kogure M. Nakamura M. Shibata Y. Katsumoto T. Biosci. Biotechnol. Biochem. 1998; 62: 718-726Crossref PubMed Scopus (84) Google Scholar, 19Knowles B.H. Knight P.J.K. Ellar D.J. Proc. R. Soc. Lond. B. 1991; 245: 31-35Crossref PubMed Scopus (89) Google Scholar). The interaction between toxin and its receptor can be complex. For example, Cry1Ac binds to two sites on the APN purified from M. sexta, and only one of these sites is also recognized by Cry1Aa and Cry1Ab (25Masson L. Lu Y.-J. Mazza A. Brousseau R. Adang M.J. J. Biol. Chem. 1995; 270: 20309-20315Crossref PubMed Scopus (136) Google Scholar). Interestingly, binding of Cry1Ac to both receptor sites is inhibited by sugars, which do not inhibit the binding of Cry1Aa and Cry1Ab (25Masson L. Lu Y.-J. Mazza A. Brousseau R. Adang M.J. J. Biol. Chem. 1995; 270: 20309-20315Crossref PubMed Scopus (136) Google Scholar). There is little information on the receptor domains involved in Cry toxin binding. In B. mori, Cry1Aa toxin binds to a conserved APN domain (26Nakanishi K. Yaoi K. Shimada N. Kadotani T. Sato R. Biochim. Biophys. Acta. 1999; 1432: 57-63Crossref PubMed Scopus (21) Google Scholar). However, the precise regions that are involved in toxin-receptor interactions, including that of the cadherin-like protein, are not known. In an attempt to identify the receptor molecules and map the receptor epitopes involved, we decided to use the phage display technique. Among several approaches used for epitope mapping, phage display has proven to be highly successful (27Cortese R. Felici F. Galfre G. Luzzago A. Monaci P. Nicosia A. Trends Biotechnol. 1994; 12: 262-267Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 28DeLeo F., Yu, L. Burritt J.B. Loetterle L.R. Bond C.B. Jesaitis A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7110-7114Crossref PubMed Scopus (134) Google Scholar, 29Demangel C. Maroun R.C. Rouyre S. Bon C. Mazié J.-C. Choumet V. Eur. J. Biochem. 2000; 267: 2345-2353Crossref PubMed Scopus (21) Google Scholar, 30Luzzago A. Felici F. Tramontano A. Pessi A. Cortese R. Gene (Amst.). 1993; 128: 51-57Crossref PubMed Scopus (246) Google Scholar, 31Szardenings M. Törnoth S. Mutulis F. Muceniece R. Keinänen K. Kuusinen A. Wikberg J.E.S. J. Biol. Chem. 1997; 272: 27943-27948Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In this study, we focused on the interaction of Cry1A toxins with brush border membrane vesicles from susceptible insects. We report here the identification of one scFv antibody whose CDR3 region shares extensive homology with an 8-amino acid region present in the cadherin-like receptors Bt-R1 and Bt-R175 from two lepidopteran insects. This 8-amino acid region competes with the binding of Cry1Ab and Cry1Aa to Bt-R1 and Bt-R175, suggesting that we identified the Cry1A toxin-binding epitopes in the cadherin-like receptor protein. Escherichia coli strains were grown in Luria broth (LB) at 37 °C either with ampicillin (100 µg/ml) or erythromycin (250 µg/ml), while Bt strains were grown in nutrient broth sporulation medium (NB) at 30 °C with or without erythromycin (7.5 µg/ml). The acrystalliferous strain 407cry − (32Lereclus D. Arantès O. Chaufaux J. Lecadet M.-M. FEMS Microbiol. Lett. 1989; 60: 211-218Google Scholar) transformed with pHT409 (33Arantès O. Lereclus D. Gene (Amst.). 1991; 108: 115-119Crossref PubMed Scopus (355) Google Scholar) harboring the cry1Aa gene or pHT315–1Ab harboring the cry1Ab gene was used for Cry1Aa and Cry1Ab production. Cry1Ac was produced from the wild type Bt strain HD73. Bt strains containing the cry1Ab,cry1Aa, and cry1Ac genes were grown for 3 days in NB. The spores and crystals were harvested and washed with buffer containing 0.01% Triton X-100, 50 mm NaCl, 50 mm Tris-HCl, pH 8.5. Crystals were isolated by sucrose gradients as previously described (34Thomas W.E. Ellar D.J. J. Cell Sci. 1983; 60: 181-197Crossref PubMed Google Scholar). These crystals were solubilized and activated by trypsin (1:50, w/w) for 2 h, and the proteins were purified by anion exchange chromatography (Q-Sepharose) as described (34Thomas W.E. Ellar D.J. J. Cell Sci. 1983; 60: 181-197Crossref PubMed Google Scholar, 35Aranda E. Sanchez J. Perferoen M. Güereca L. Bravo A. J. Invertebr. Pathol. 1996; 68: 203-212Crossref PubMed Scopus (103) Google Scholar). The purified toxins were concentrated in dialysis bags (Spectra/Por, cut-off 12–14 kDa; Fisher) covered with polyethylene glycol 8000, dialyzed against 1000 volumes of buffer A (150 mm N-methylglucamine chloride, 10 mm HEPES, pH 8), and stored at 4 °C until used. Toxins were apparently homogeneous as determined by SDS-PAGE and silver staining. The Nissim synthetic phage-antibody library used in this work was kindly provided by the Cambridge Center for Protein Engineering (Cambridge, UK). This library, with a diversity of 1 × 108 clones, contains a diverse repertoire of in vitro rearranged VH genes containing a random VH-CDR3 of 4–12 amino acid residues in length (36Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Lidgley C. Lane D. Winter G. EMBO J. 1994; 13: 692-698Crossref PubMed Scopus (528) Google Scholar). Cry1Ab-binding phages were isolated by panning using immunotubes (Nunc), which were coated with (100 µg/ml) Cry1Ab toxin overnight at room temperature. After each round of selection, individual clones were analyzed for their ability to bind Cry1Ab by enzyme-linked immunosorbent assay. The helper phage VCS-M13 (Stratagene) was used to rescue phages from individual colonies of infected E. coliTG-1. Expression of soluble fragments from single infected E. coli HB2151 colonies (36Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Lidgley C. Lane D. Winter G. EMBO J. 1994; 13: 692-698Crossref PubMed Scopus (528) Google Scholar, 37Hoogenboom H.R. Winter G. J. Mol. Biol. 1992; 227: 381-388Crossref PubMed Scopus (391) Google Scholar, 38Marks J.D. Hoogenboom H.R. Bonnert T.P. MacCafferty J. Griffiths A.D. Winter G. J. Mol. Biol. 1991; 222: 581-597Crossref PubMed Scopus (1437) Google Scholar) was induced by isopropyl thiogalactoside. Bacterial supernatants containing phage or scFv fragments were screened for toxin binding by enzyme-linked immunosorbent assay. DNA fingerprinting was performed by amplifying the scFv insert using primers LMB3 (5′-CAGGAAACAGCTATGAC) and fd-SEQ1 (5′-GAATTTTCTGTATGAGG) followed by digestion with the frequently cutting enzyme BstNI as described (37Hoogenboom H.R. Winter G. J. Mol. Biol. 1992; 227: 381-388Crossref PubMed Scopus (391) Google Scholar). CDR3 sequence was determined using the primer CDRFOR (5′-CAGGGTACCTTGGCCCCA) (38Marks J.D. Hoogenboom H.R. Bonnert T.P. MacCafferty J. Griffiths A.D. Winter G. J. Mol. Biol. 1991; 222: 581-597Crossref PubMed Scopus (1437) Google Scholar). For purification of scFv molecules, scFv genes were subcloned into the pSyn vector (38Marks J.D. Hoogenboom H.R. Bonnert T.P. MacCafferty J. Griffiths A.D. Winter G. J. Mol. Biol. 1991; 222: 581-597Crossref PubMed Scopus (1437) Google Scholar) and used to transform E. coli TG1. scFv fragments were purified to homogeneity as follows. Selected clones were cultured at 37 °C in 2× TY (supplemented with 100 µg/ml ampicillin and 0.1% glucose) until they reached an OD of 0.7 at 600 nm. Production of soluble scFv was induced by the addition of 0.5 mmisopropyl thiogalactoside to the culture and grown for 4 h at 25 °C. The scFv was collected from the periplasm. Soluble periplasmic extracts were obtained by osmotic shock at 4 °C using lysis buffer containing 200 mg/ml sucrose, 1 mm EDTA, 300 mm Tris-HCl, pH 8. The supernatant was applied to a nickel-agarose column, which was washed with PBS, and the scFv was eluted with 2 ml of 250 mm imidazole, 0.2% azide in PBS. DomI and DomII-III-H6 of the Cry1Ab toxin were individually expressed in BL21 E. coli cells as described (39Flores H. Soberón X. Sánchez J. Bravo A. FEBS Lett. 1997; 414: 313-318Crossref PubMed Scopus (23) Google Scholar). 2A. Bravo, R. Meza, and A. Lorence, unpublished results. Briefly, an overnight culture of pDomII-III-H6 (or pDomI) transformed cells was grown at 37 °C in LB medium (200 µg/ml ampicillin). This culture was used to inoculate 100 ml of LB medium (1:100 dilution). The cells were grown to an OD of 0.5–0.6 and induced with 1 mm isopropyl thiogalactoside. After 3 h of growth, the cells were centrifuged and suspended in 3 ml of buffer A (50 mm NaSO4,300 mm NaCl, pH 8). Cells were then sonicated on ice (two 1-min bursts) and centrifuged (10 min at 12,000 × g). Soluble proteins (10 µg) were separated by 10% SDS-PAGE, and Western blot analysis was performed as described (35Aranda E. Sanchez J. Perferoen M. Güereca L. Bravo A. J. Invertebr. Pathol. 1996; 68: 203-212Crossref PubMed Scopus (103) Google Scholar), using bacterial supernatants containing phage or scFv fragments. For scFv fragments, a c-Myc antibody (Sigma) (1:1000 dilution) was used, followed by incubation with a secondary goat anti-mouse antibody conjugated with peroxidase (Sigma) (1:1000 dilution). For clone M13–19, an anti-M13 antibody conjugated to peroxidase (Sigma) (1:1000 dilution) was utilized as described (34Thomas W.E. Ellar D.J. J. Cell Sci. 1983; 60: 181-197Crossref PubMed Google Scholar, 35Aranda E. Sanchez J. Perferoen M. Güereca L. Bravo A. J. Invertebr. Pathol. 1996; 68: 203-212Crossref PubMed Scopus (103) Google Scholar). Blots were visualized using luminol (ECL; Amersham Pharmacia Biotech). M. sexta eggs were kindly supplied by Dr. Jorge Ibarra (CINVESTAV, Irapuato), and B. mori eggs were obtained from Carolina Biological Supply Co. M. sexta andB. mori larvae were reared on an artificial diet and fresh mulberry leaves, respectively. BBMVs from fifth instar M. sexta or B. mori larvae were prepared as reported (41Wolfersberger M. Lüthy P. Maurer A. Parenti P. Sacchi F.V. Giordana B. Hanozet G.M. Comp. Biochem. Physiol. 1987; 86A: 301-308Crossref Scopus (545) Google Scholar) except that neomycin sulfate (2.4 µg/ml) was included in the buffer (300 mm mannitol, 2 mm dithiothreitol, 5 mm EGTA, 1 mm EDTA, 0.1 mmphenylmethylsulfonyl fluoride, 150 µg ml−1 pepstatin A, 100 µg ml−1 leupeptin, 1 µg ml−1 soybean trypsin inhibitor, 10 mm HEPES-HCl, pH 7.4). Binding and competition analyses of Cry1Aa-c toxins to M. sexta andB. mori BBMV were performed as previously described (35Aranda E. Sanchez J. Perferoen M. Güereca L. Bravo A. J. Invertebr. Pathol. 1996; 68: 203-212Crossref PubMed Scopus (103) Google Scholar). Amino acid sequences of synthetic peptides used for competition experiments were the following: CDR3–73 (RITQTTNRAA), BtR1-CRY (HITDTNNKAA), BtR175-CRY1 (QIIDTNNKAA), BtR175-CRY2 (LDETTNVLAA), and PepL1 (TDAHRGEYYW). Toxins were biotinylated using biotinyl-N-hydroxysuccinimide ester (Amersham Pharmacia Biotech), and binding analyses were performed in 100 µl of binding buffer (PBS, 0.1% (w/v) BSA, 0.1% (v/v) Tween 20, pH 7.6). Ten micrograms of BBMV protein were incubated with 10 nmbiotinylated toxin, and the unbound toxin was removed by centrifugation for 10 min at 14,000 × g. The pellet containing BBMV and the bound biotinylated toxin was suspended in 100 µl of binding buffer and washed twice. Finally, the BBMVs were suspended in 10 µl of PBS, pH 7.6, and an equal volume of 2× sample loading buffer (0.125m Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.01% bromphenol blue) was added. The samples were separated by SDS-polyacrylamide gels and electrotransferred to nitrocellulose membranes. The biotinylated protein was visualized by incubating with streptavidin-peroxidase conjugate (1:4000 dilution) for 1 h, followed by luminol (ECL; Amersham Pharmacia Biotech). For competition experiments, biotinylated Cry1A toxins were incubated with different concentrations of scFvs or peptides in PBS for 1 h at room temperature before incubating toxin with BBMV. Protein blot analysis of BBMV preparations was performed as described previously (35Aranda E. Sanchez J. Perferoen M. Güereca L. Bravo A. J. Invertebr. Pathol. 1996; 68: 203-212Crossref PubMed Scopus (103) Google Scholar, 41Wolfersberger M. Lüthy P. Maurer A. Parenti P. Sacchi F.V. Giordana B. Hanozet G.M. Comp. Biochem. Physiol. 1987; 86A: 301-308Crossref Scopus (545) Google Scholar). Ten micrograms of BBMV protein were separated by 9% SDS-PAGE and electrotransferred to nitrocellulose membranes. After blocking, the membranes were incubated for 2 h with 10 nmbiotinylated Cry1A toxins. Unbound toxin was removed by washing the membrane three times with washing buffer for 10 min, and the bound toxin was identified by incubation with streptavidin-peroxidase conjugate (1:5000) for 1 h and visualized using luminol (ECL;Amersham Pharmacia Biotech). For competition experiments, biotinylated Cry1A toxins were incubated with different concentrations of scFvs or peptides in washing buffer (0.1% Tween 20, 0.2% BSA in PBS) for 1 h at room temperature before incubating toxin with nitrocellulose membranes. All surface plasmon resonance (SPR) measurements were performed using a Biacore X and CM5 sensor chips (Biacore). HBS-P buffer (10 mm HEPES, pH 7.4, 0.15 m NaCl, 3 mmEDTA, 005% surfactant P20) was used throughout the analyses. The ligand, scFv73 (30 kDa, apparently homogeneous based on SDS-PAGE) at a concentration of 25 µg/ml in 20 mm ammonium acetate, pH 5, buffer, was immobilized on flow cell 2 using a standard amine-coupling kit (Biacore) at densities of less than 150 response units. The surfaces of both flow cells were activated for 5 min at a flow rate of 10 µl/min. Following ligand immobilization on flow cell 2, both flow cells were blocked with a 5-min injection of 1m ethanolamine at a flow rate of 10 µl/min. The analytes (65 kDa, apparently homogenous based on SDS-PAGE) were injected over both flow cells at a flow rate of 30 µl/min. The complex was allowed to associate and dissociate for 120 and 180 s, respectively. The surfaces were regenerated with a 1-min injection of 1 mmHCl. Triplicate injections of each toxin concentration were injected in random order over both surfaces, and the responses were corrected by double referencing (42Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (656) Google Scholar). The data were fitted using global analysis software available within Biaevaluation 3.1 (Biacore). Competition experiments were performed by injection of a 10- and 200-fold molar excess of scFv in combination with Cry1Ab and Cry1Aa toxins. Carbohydrate inhibition studies with GalNAc were carried out using 600 nm Cry1Ac and 20 µm GalNAc. As an additional control, we immobilized a non-Cry1A-binding scFv4E that was obtained by panning against a different antigen onto flow cell 1 at similar levels as scFv73. Various concentrations of toxin were injected over both flow cells, and the response curve on flow cell 1 was subtracted from flow cell 2. Using this control flow cell configuration, identical Cry1Ab binding curves were obtained compared with using the ethanolamine-blocked control surface. Bioassays were performed with M. sexta neonate larvae using surface-treated food with 9 ng/cm2 as reported (5Bravo A. Jansens S. Peferoen M. J. Invertebr. Pathol. 1992; 60: 237-246Crossref Scopus (109) Google Scholar), and mortality was recorded after 7 days. A library of 108 human single chain antibody fragments (scFv) with variability in the CDR3 region (5–12 amino acids) (36Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Lidgley C. Lane D. Winter G. EMBO J. 1994; 13: 692-698Crossref PubMed Scopus (528) Google Scholar) was used to select a population of phages that bound Cry1Ab toxin. After eight rounds of panning, 98% of the M13 phages bound Cry1Ab (data not shown). To characterize the phages isolated, we amplified the variable regions by PCR and digested the products with BstNI restriction enzyme. Analysis of 50 phage clones showed three different restriction patterns (data not shown). One of these patterns was found in 48 of the clones analyzed (representative clone scFv45), while the other two patterns were each represented by one clone. DNA sequence analysis of the CDR3 region was determined for 10 clones of the most abundant restriction pattern (including scFv45) and also for the two clones representing the other two unique restriction patterns (scFv19 and scFv73). Three different amino acid sequences were present in the CDR3 regions of the clones analyzed (scFv19, RTSPRLTPKHR; scFv73, ITQTTNR; scFv45, NPRIPP). We used Western blot analysis to determine which Cry1Ab toxin domain bound the three scFv antibodies. This analysis was performed using the scFv antibodies against membrane blots containing protein extracts fromE. coli expressing either Cry1Ab domain I or domains II-III. Fig. 1 shows that the three scFv clones recognized the 44-kDa domain II-III polypeptide but not the 30-kDa domain I polypeptide. To analyze whether domain II or III was recognized by these scFv proteins, we determined if the three M13-scFv phages bound Cry1Ac toxin, since this toxin shares 98% identity with Cry1Ab toxin in domain II but only 38% identity in domain III. Enzyme-linked immunosorbent assay binding analysis showed that the three scFv antibodies also recognized Cry1Ac toxin (data not shown), suggesting that the three scFv fragments bound to domain II of Cry1Ab toxin. The three anti-Cry1Ab scFv genes were subcloned into a plasmid to incorporate a hexahistidine tag and then expressed and purified from E. coli. Only scFv73 and scFv45 were produced in E. coli in high quantities, and scFv19 was therefore not analyzed further. To determine if the selected scFv antibodies could compete with the binding of Cry1Ab toxin to its receptor, we performed two different binding assays. In the first protocol, a qualitative binding assay, biotinylated Cry1Ab toxin was incubated in solution with BBMV. The bound toxin was visualized following SDS-PAGE and electrotransfer of the proteins to nitrocellulose membranes. Fig.2 A shows that both scFv antibodies compete with the binding of Cry1Ab toxin to M. sexta BBMV, although scFv73 competes more efficiently than scFv45 (Fig. 2 A, lanes 4 and 6). The second protocol, toxin overlay assays, allows the identification of BBMV proteins that interact with Cry1Ab. BBMV proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. Then biotinylated Cry1Ab toxin was incubated with the membranes, and the proteins that bound the toxin were detected with streptavidin coupled to peroxidase. Fig. 2 B shows that both the 120-kDa aminopeptidase (APN) (13Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbi" @default.
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• W2095777724 title "Mapping the Epitope in Cadherin-like Receptors Involved inBacillus thuringiensis Cry1A Toxin Interaction Using Phage Display" @default.
• W2095777724 cites W123753296 @default.
• W2095777724 cites W1489538658 @default.
• W2095777724 cites W1549258100 @default.
• W2095777724 cites W1593307737 @default.
• W2095777724 cites W1965946471 @default.
• W2095777724 cites W1976908016 @default.
• W2095777724 cites W19835385 @default.
• W2095777724 cites W1986863534 @default.
• W2095777724 cites W1990002364 @default.
• W2095777724 cites W1994225738 @default.
• W2095777724 cites W1994501723 @default.
• W2095777724 cites W1995139888 @default.
• W2095777724 cites W1996740379 @default.
• W2095777724 cites W2007879139 @default.
• W2095777724 cites W2008726255 @default.
• W2095777724 cites W2010801138 @default.
• W2095777724 cites W2014106512 @default.
• W2095777724 cites W2024018480 @default.
• W2095777724 cites W2025967034 @default.
• W2095777724 cites W2026920317 @default.
• W2095777724 cites W2036072604 @default.
• W2095777724 cites W2041716585 @default.
• W2095777724 cites W2045821897 @default.
• W2095777724 cites W2059543717 @default.
• W2095777724 cites W2060579576 @default.
• W2095777724 cites W2067631233 @default.
• W2095777724 cites W2070070968 @default.
• W2095777724 cites W2070207092 @default.
• W2095777724 cites W2072530473 @default.
• W2095777724 cites W2073427943 @default.
• W2095777724 cites W2078675196 @default.
• W2095777724 cites W2081319380 @default.
• W2095777724 cites W2081335753 @default.
• W2095777724 cites W2088096803 @default.
• W2095777724 cites W2090103138 @default.
• W2095777724 cites W2092234562 @default.
• W2095777724 cites W2103890327 @default.
• W2095777724 cites W2103967018 @default.
• W2095777724 cites W2107658967 @default.
• W2095777724 cites W2107847732 @default.
• W2095777724 cites W2121016086 @default.
• W2095777724 cites W2121484976 @default.
• W2095777724 cites W2148358473 @default.
• W2095777724 cites W2157549022 @default.
• W2095777724 cites W4239011189 @default.
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