Abstract
Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 105 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arias JL, Unciti-Broceta JD, Maceira J, Del Castillo T, Hernández-Quero J, Magez S, Soriano M, García-Salcedo JA (2015) Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis. J Control Release 197:190–198. doi:10.1016/j.jconrel.2014.11.002
Arnaout R, Lee W, Cahill P, Honan T, Sparrow T, Weiand M, Chad Nusbaum C, Rajewsky K, Koralov SB (2011) High-resolution description of antibody heavy-chain repertoires in humans. PLoS One 6(8):e22365. doi:10.1371/journal.pone.0022365
Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH (2005) Evolving strategies for enzyme engineering. Curr Opin Struct Biol 15(4):447–452. doi:10.1016/j.sbi.2005.06.004
Boder ET, Wittrup KD (2000) [25] Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:430–444. doi:10.1016/S0076-6879(00)28410-3
Bowie JU, Lüthy R, Eisenberg DA (1991) Method to identify protein sequences that fold into a known three-dimensional structure. Science 253(5016):164–170. doi:10.1126/science.1853201
Bravo A, Soberón M (2008) How to cope with insect resistance to Bt toxins? Trends Biotechnol 26(10):573–579. doi:10.1016/j.tibtech.2008.06.005
Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Molec Biol 41(7):423–431. doi:10.1016/j.ibmb.2011.02.006
Brückner A, Polge C, Lentze N, Auerbach D, Schlattner U (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10(6):2763–2788. doi:10.3390/ijms10062763
Cauerhff A, Goldbaum FA, Braden BC (2004) Structural mechanism for affinity maturation of an anti-lysozyme antibody. Proc Natl Acad Sci U S A 101(10):3539–3544. doi:10.1073/pnas.0400060101
Chowdhury PS, Pastan I (1999) Improving antibody affinity by mimicking somatic hypermutation in vitro. Nat Biotechnol 17(6):568–572. doi:10.1038/9872
Clark LA, Ganesan S, Papp S, van Vlijmen HW (2006) Trends in antibody sequence changes during the somatic hypermutation process. J Immunol 177(1):333–340. doi:10.4049/jimmunol.177.1.333
De Meyer T, Muyldermans S, Depicker A (2014) Nanobody-based products as research and diagnostic tools. Trends Biotechnol 32(5):263–270. doi:10.1016/j.tibtech.2014.03.001
Dong S, Zhang CZ, Zhang X, Liu Y, Zhong J, Xie Y, Xu CX, Ding Y, Zhang LQ, Liu XJ (2016) Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins using designed polypeptide as hapten. Anal Chem 88:7023–7032. doi:10.1021/acs.analchem.6b00429
Eteshola E (2010) Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display. J Immunol Methods 358(1):104–110. doi:10.1016/j.jim.2010.04.003
Eyer L, Hruska K (2012) Single-domain antibody fragments derived from heavy-chain antibodies: a review. Vet Med 57(9):439–513 https://www.researchgate.net/publication/285682971
Fang T, Duarte JN, Ling J, Li Z, Guzman JS, Ploegh HL (2016) Structurally defined αMHC-II nanobody-drug conjugates: a therapeutic and imaging system for B-cell lymphoma. Angew Chem Int Ed 55(7):2416–2420. doi:10.1002/anie. 201509432
Garet E, Cabado AG, Vieites JM, González-Fernández Á (2010) Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: palytoxin. Toxicon 55(8):1519–1526. doi:10.1016/j.toxicon.2010.03.005
Gibbs WW (2005) Nanobodies. Sci Am 293(2):78–83. doi:10.1038/scientificamerican0805-78
Gruère G P, Rao S R (2007) A review of international labeling policies of genetically modified food to evaluate India’s proposed rule. http://agbioforum.org/v10n1/v10n1a06-gruere.htm
Hayashi N, Welschof M, Zewe M, Braunagel M, Dübel S, Breitling F, Little M (1994) Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR. BioTechniques 17(2):310–312 314-315.https://www.ncbi.nlm.nih.gov/pubmed/7980934
Höfte, H, Whiteley, HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53(2):242–255
Jermutus L, Honegger A, Schwesinger F, Hanes J, Pluckthun A (2001) Tailoring in vitro evolution for protein affinity or stability. Proc Natl Acad Sci 98(1):75–80
Jiao L, Xu X, Liu Y, Zhang X, Liang Y, Liu X (2015) Screening and identification of humanized single domain antibodies (sdAbs) against Bacillus thuringiensis Cry1Ab toxin. Chin J Food Sci Accepted
Korpimäki T, Brockmann EC, Kuronen O, Saraste M, Lamminmäki U, Tuomola M (2004) Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level. J Agric Food Chem 52(1):40–47. doi:10.1021/jf034951i
Korpimäki T, Rosenberg J, Virtanen P, Karskela T, Lamminmäki U, Tuomola M, Vehniäinen M, Saviranta P (2002) Improving broad specificity hapten recognition with protein engineering. J Agr Food Chem 50(15):4194–4201. doi:10.1021/jf0200624
Korpimäki T, Rosenberg J, Virtanen P, Lamminmäki U, Tuomola M, Saviranta P (2003) Further improvement of broad specificity hapten recognition with protein engineering. Protein Eng 16(1):37–46. doi:10.1093/proeng/gzg010
Kuroda D, Shirai H, Jacobson MP, Nakamura H (2012) Computer-aided antibody design. PEDS 25(10):507–521. doi:10.1093/protein/gzs024
Laeremans T, Henegouwen P M V B E, Silence K, Vaeck M (2011) U.S. Patent Application No. 13/016,709.
Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8(4):477–486. doi:10.1007/BF00228148
Leivo J, Chappuis C, Lamminmäki U, Lövgren T, Vehniäinen M (2011) Engineering of a broad-specificity antibody: detection of eight fluoroquinolone antibiotics simultaneously. Anal Biochem 409(1):14–21. doi:10.1016/j.ab.2010.09.041
Lorenzen N, Olesen NJ, Jorgensen PE (1990) Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. J Gen Virol 71(3):561–567. doi:10.1099/0022-1317-71-3-561
McDonnell B, Hearty S, Finlay WJ, O’Kennedy R (2011) A high-affinity recombinant antibody permits rapid and sensitive direct detection of myeloperoxidase. Anal Biochem 410(1):1–6. doi:10.1016/j.ab.2010.09.039
Morrison KL, Weiss GA (2001) Combinatorial alanine-scanning. Curr Opin Chem Biol 5(3):302–307. doi:10.1016/S1367-5931(00)00206-4
National Academies of Sciences, Engineering, and Medicine (2016) Genetically engineered crops: experiences and prospects. The National Academies Press, Washington, DC. doi:10.17226/23395
Park SG, Jung YJ, Lee YY, Yang CM, Kim IJ, Chung JH, Kim IS, Lee YJ, Park SJ, Lee JN, Seo SK (2006) Improvement of neutralizing activity of human scFv antibodies against hepatitis B virus binding using CDR3 VH mutant library. Viral Immunol 19(1):115–123. doi:10.1089/vim.2006.19.115
Qiu T, Xiao H, Zhang Q, Qiu J, Yang Y, Wu D, Cao Z, Zhu R (2015) Proteochemometric modeling of the antigen-antibody interaction: new fingerprints for antigen, antibody and epitope-paratope interaction. PLoS One 10(4):e0122416. doi:10.1371/journal.pone.0122416
Raddadi N, Crotti E, Rolli E, Marasco R, Fava F, Daffonchio D (2012) The most important Bacillus species in biotechnology. In: Bacillus thuringiensis Biotechnology (329-345). Springer, Netherlands. doi:10.1007/978-94-007-3021-2_17
Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2(4):891–903. doi:10.1038/nprot.2007.72
Roda A, Mirasoli M, Guardigli M, Michelini E, Simoni P, Magliulo M (2006) Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize. Anal Bioanal Chem 384(6):1269–1275. doi:10.1007/s00216-006-0308-6
Schmitz U, Versmold A, Kaufmann P, Frank HG (2000) Phage display: a molecular tool for the generation of antibodies-a review. Placenta 21:S106–S112. doi:10.1053/plac.1999.0511
Sevy AM, Meiler J (2014) Antibodies: computer-aided prediction of structure and design of function. Microbiol Spectr 2(6):1–14. doi:10.1128/microbiolspec.AID-0024-2014
Shao ES, Lin L, Shi P, Liu SJ, Guan X, Huang ZP (2014) Impact of three exposed loops in domain II of Cry1Ab toxin of Bacillus thringiensis on its insecticidal activity. J Agric Biotechnol 22(11):1357–1366 http://manu02.magtech.com.cn/Jwk_ny/CN/Y2014/V22/I11/1357
Sheedy C, MacKenzie CR, Hall JC (2007) Isolation and affinity maturation of hapten-specific antibodies. Biotechnol Adv 25(4):333–352. doi:10.1016/j.biotechadv.2007.02.003
Steidl S, Ratsch O, Brocks B, Dürr M, Thomassen-Wolf E (2008) In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification. Mol Immunol 46(1):135–144. doi:10.1016/j.molimm.2008.07.013
Wang P, Li G, Yan J, Hu Y, Zhang C, Liu X, Wan Y (2014) Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin. Toxicon 92:186–192. doi:10.1016/j.toxicon.2014.10.024
Wang Y, Zhang X, Zhang C, Liu Y, Liu X (2012) Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries. Toxicon 60(7):1290–1297. doi:10.1016/j.toxicon.2012.08.014
Yau KY, Dubuc G, Li S, Hirama T, MacKenzie CR, Jermutus L, Hall JC, Tanha J (2005) Affinity maturation of a VHH by mutational hotspot randomization. J Immunol Methods 297(1):213–224. doi:10.1016/j.jim.2004.12.005
Zhang X, Liu Y, Zhang C, Wang Y, Xu C, Liu X (2012) Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin. Ecotox Environ Safe 81:84–90. doi:10.1016/j.ecoenv.2012.04.021
Zhang X, Xu C, Zhang C, Liu Y, Xie Y, Liu X (2014) Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naive mouse phage displayed library. Toxicon 81:13–22. doi:10.1016/j.toxicon.2014.01.010
Zhou Q, Li G, Zhang Y, Zhu M, Wan Y, Shen Y (2016) Highly selective and sensitive electrochemical immunoassay of Cry1C using nanobody and π–π stacked graphene oxide/thionine assembly. Anal Chem 88(19):9830–9836. doi:10.1021/acs.analchem.6b02945
Acknowledgements
This study was sponsored by National Natural Science Foundation of China (31630061).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical statement
This article does not contain any studies with human participants or animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
Jiao, L., Liu, Y., Zhang, X. et al. Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins. Appl Microbiol Biotechnol 101, 6071–6082 (2017). https://doi.org/10.1007/s00253-017-8347-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8347-9