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Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins

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Abstract

Antibodies and antibody derived fragments are excellent tools for the detection and purification of proteins. However, only few antibodies targeting Arabidopsis seed proteins are currently available. Here, we evaluate the process to make antibody libraries against crude protein extracts and more particularly to generate a VHH phage library against native Arabidopsis thaliana seed proteins. After immunising a dromedary with a crude Arabidopsis seed extract, we cloned the single-domain antigen-binding fragments from their heavy-chain only antibodies in a phage display vector and selected nanobodies (VHHs) against native Arabidopsis seed proteins. For 16 VHHs, the corresponding antigens were identified by affinity purification and MS/MS analysis. They were shown to bind the major Arabidopsis seed storage proteins albumin and globulin (14 to albumin and 2 to globulin). All 16 VHHs were suitable primary reagents for the detection of the Arabidopsis seed storage proteins by ELISA. Furthermore, several of the anti-albumin VHHs were used successfully for storage protein localisation via electron microscopy. The easy cloning, selection and production, together with the demonstrated functionality and applicability, strongly suggest that the VHH antibody format will play a more prominent role in future protein research, in particular for the study of native proteins.

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References

  • Abbady AQ, Al-Mariri A, Zarkawi M, Al-Assad A, Muyldermans S (2011) Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel. Vet Immunol Immunopathol 142(1–2):49–56. doi:10.1016/j.vetimm.2011.04.004

    Article  PubMed  CAS  Google Scholar 

  • Abbady AQ, Al-Daoude A, Al-Mariri A, Zarkawi M, Muyldermans S (2012) Chaperonin GroEL a Brucella immunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies. Vet Immunol Immunopathol 146(3–4):254–263. doi:10.1016/j.vetimm.2012.01.015

    Article  PubMed  CAS  Google Scholar 

  • Adachi M, Kanamori J, Masuda T, Yagasaki K, Kitamura K, Mikami B, Utsumi S (2003) Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer. Proc Natl Acad Sci USA 100(12):7395–7400. doi:10.1073/pnas.0832158100

    Article  PubMed  CAS  Google Scholar 

  • Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2005) Prokaryotic expression of antibodies. Cancer Metastasis Rev 24(4):501–519. doi:10.1007/s10555-005-6193-1

    Article  PubMed  Google Scholar 

  • Baens M, Noels H, Broeckx V, Hagens S, Fevery S, Billiau AD, Vankelecom H, Marynen P (2006) The dark side of EGFP: defective polyubiquitination. PLoS One 1(1):e54. doi:10.1371/journal.pone.0000054

    Article  PubMed  Google Scholar 

  • Baerenfaller K, Grossmann J, Grobei MA, Hull R, Hirsch-Hoffmann M, Yalovsky S, Zimmermann P, Grossniklaus U, Gruissem W, Baginsky S (2008) Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics. Science 320(5878):938–941. doi:10.1126/science.1157956

    Article  PubMed  CAS  Google Scholar 

  • Bernal AJ, Willats WGT (2004) Plant science in the age of phage. Trends Plant Sci 9(10):465–468. doi:10.1016/j.tplants.2004.08.004

    Article  PubMed  CAS  Google Scholar 

  • Claeys M, Vanhecke D, Couvreur M, Tytgat T, Coomans A, Borgonie G (2004) High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy. Nematology 6(3):319–327. doi:10.1163/1568541042360564

    Article  Google Scholar 

  • De Buck S, Virdi V, De Meyer T, De Wilde K, Piron R, Nolf J, Van Lerberge E, De Paepe A, Depicker A (2012) Production of camel-like antibodies in plants. Methods Mol Biol 911:305–324

    PubMed  Google Scholar 

  • De Clercq A, Vandewiele M, De Rycke R, Van Damme J, Van Montagu M, Krebbers E, Vandekerckhove J (1990) Expression and processing of an Arabidopsis 2S albumin in transgenic tobacco. Plant Physiol 92(4):899–907. doi:10.1104/pp.92.4.899

    Article  PubMed  Google Scholar 

  • De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne R, Muyldermans S, Wyns L (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci USA 103(12):4586–4591. doi:10.1073/pnas.0505379103

    Article  PubMed  Google Scholar 

  • Deckers N, Saerens D, Kanobana K, Conrath K, Victor B, Wernery U, Vercruysse J, Muyldermans S, Dorny P (2009) Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis. Int J Parasit 39(5):625–633. doi:10.1016/j.ijpara.2008.10.012

    Article  CAS  Google Scholar 

  • Dübel S, Stoevesandt O, Taussig MJ, Hust M (2010) Generating recombinant antibodies to the complete human proteome. Trends Biotechnol 28(7):333–339. doi:10.1016/j.tibtech.2010.05.001

    Article  PubMed  Google Scholar 

  • Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, Muyldermans S, Wyns L, Matagne A (2002) Single-domain antibody fragments with high conformational stability. Protein Sci 11(3):500–515. doi:10.1110/ps.34602

    Article  PubMed  CAS  Google Scholar 

  • Eeckhout D, De Clercq A, Van De Slijke E, Van Leene J, Stals H, Casteels P, Persiau G, Vercammen D, Van Breusegem F, Zabeau M, Inzé D, Jespers L, Depicker A, De Jaeger G (2004) A technology platform for the fast production of monoclonal recombinant antibodies against plant proteins and peptides. J Immunol Methods 294(1–2):181–187. doi:10.1016/j.jim.2004.08.006

    Article  PubMed  CAS  Google Scholar 

  • Freydank A-C, Brandt W, Dräger B (2008) Protein structure modeling indicates hexahistidine-tag interference with enzyme activity. Proteins 72(1):173–183. doi:10.1002/prot.21905

    Article  PubMed  CAS  Google Scholar 

  • Fujiwara T, Nambara E, Yamagishi K, Goto DB, Naito S (2002) Storage proteins. In: Somerville CR, Meyerowitz EM (eds) The arabidopsis book, vol 1. American Society of Plant Physiologists, Rockville, pp 1–12. doi:10.1199/tab.0020

  • Ghassabeh GH, Saerens D, Muyldermans S (2010) Isolation of antigen-specific nanobodies. In: Kontermann R, Dübel S (eds) Antibody engineering, vol 2. Springer, Berlin, pp 251–266

    Chapter  Google Scholar 

  • Goossens A, Dillen W, De Clercq J, Van Montagu M, Angenon G (1999) The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants. Plant Physiol 120(4):1095–1104

    Article  PubMed  CAS  Google Scholar 

  • Gorlani A, de Haard H, Verrips T (2012) Expression of VHHs in Saccharomyces cerevisiae. Methods Mol Biol 911:277–286. doi:10.1007/978-1-61779-968-6_17

    PubMed  CAS  Google Scholar 

  • Groot AJ, El Khattabi M, Sachs N, van der Groep P, van der Wall E, van Diest PJ, Sonnenberg A, Verrips CT, Vooijs M (2009) Reverse proteomic antibody screening identifies anti adhesive VHH targeting VLA-3. Mol Immunol 46(10):2022–2028. doi:10.1016/j.molimm.2009.03.002

    Article  PubMed  CAS  Google Scholar 

  • Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363(6428):446–448. doi:10.1038/363446a0

    Article  PubMed  CAS  Google Scholar 

  • Hara-Nishimura I, Shimada T, Hatano K, Takeuchi Y, Nishimura M (1998) Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles. Plant Cell 10(5):825–836

    PubMed  CAS  Google Scholar 

  • Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77(1):13–22. doi:10.1007/s00253-007-1142-2

    Article  PubMed  CAS  Google Scholar 

  • Herman EM, Larkins BA (1999) Protein storage bodies and vacuoles. Plant Cell 11(4):601–613

    PubMed  CAS  Google Scholar 

  • Higashi Y, Hirai MY, Fujiwara T, Naito S, Noji M, Saito K (2006) Proteomic and transcriptomic analysis of Arabidopsis seeds: molecular evidence for successive processing of seed proteins and its implication in the stress response to sulfur nutrition. Plant J 48(4):557–571. doi:10.1111/j.1365-313X.2006.02900.x

    Article  PubMed  CAS  Google Scholar 

  • Hmila I, Abdallah RBA-B, Saerens D, Benlasfar Z, Conrath K, Ayeb ME, Muyldermans S, Bouhaouala-Zahar B (2008) VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’. Mol Immunol 45(14):3847–3856. doi:10.1016/j.molimm.2008.04.011

    Article  PubMed  CAS  Google Scholar 

  • Hu W-G, Yin J, Jager S, Wong C, Fulton C, Rayner GA, Aw C, Fisher GR, Dai X, Nagata LP (2008) A novel approach to development of monoclonal antibodies using native antigen for immunization and recombinant antigen for screening. Hybridoma 27(4):307–311. doi:10.1089/hyb.2008.0011

    Article  PubMed  Google Scholar 

  • Jenkins JA, Griffiths-Jones S, Shewry PR, Breiteneder H, Mills ENC (2005) Structural relatedness of plant food allergens with specific reference to cross-reactive allergens: an in silico analysis. J Allergy Clin Immunol 115(1):163–170. doi:10.1016/j.jaci.2004.10.026

    Article  PubMed  CAS  Google Scholar 

  • Jensen BC, Swigart PM, Simpson PC (2009) Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. Naunyn-Schmiedeberg’s Arch Pharmacol 379(4):409–412. doi:10.1007/s00210-008-0368-6

    Article  CAS  Google Scholar 

  • Krebbers E, Herdies L, De Clercq A, Seurinck J, Leemans J, Van Damme J, Segura M, Gheysen G, Van Montagu M, Vandekerckhove J (1988) Determination of the processing sites of an Arabidopsis 2S albumin and characterization of the complete gene family. Plant Physiol 87(4):859–866. doi:10.1104/pp.87.4.859

    Article  PubMed  CAS  Google Scholar 

  • Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Hölzer W, De Genst E, Wyns L, Muyldermans S (1998) Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17(13):3512–3520. doi:10.1093/emboj/17.13.3512

    Article  PubMed  CAS  Google Scholar 

  • Leduc C, Si S, Gautier J, Soto-Ribeiro M, Wehrle-Haller B, Gautreau A, Giannone G, Cognet L, Lounis B (2013) A highly specific gold nanoprobe for live-cell single-molecule imaging. Nano Lett 13(4):1489–1494. doi:10.1021/nl304561g

    PubMed  CAS  Google Scholar 

  • Moutel S, El Marjou A, Vielemeyer O, Nizak C, Benaroch P, Dübel S, Perez F (2009) A multi-Fc-species system for recombinant antibody production. BMC Biotechnol 9:14. doi:10.1186/1472-6750-9-14

    Article  PubMed  Google Scholar 

  • Müntz K (1998) Deposition of storage proteins. Plant Mol Biol 38(1–2):77–99. doi:10.1007/978-94-011-5298-3_4

    Article  PubMed  Google Scholar 

  • Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem. doi:10.1146/annurev-biochem-063011-092449

  • Nilsson P, Paavilainen L, Larsson K, Ödling J, Sundberg M, Andersson A-C, Kampf C, Persson A, Al-Khalili Szigyarto C, Ottosson J, Björling E, Hober S, Wernérus H, Wester K, Pontén F, Uhlen M (2005) Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling. Proteomics 5(17):4327–4337. doi:10.1002/pmic.200500072

    Article  PubMed  CAS  Google Scholar 

  • Pang PP, Pruitt RE, Meyerowitz EM (1988) Molecular cloning, genomic organization, expression and evolution of 12S seed storage protein genes of Arabidopsis thaliana. Plant Mol Biol 11(6):805–820. doi:10.1007/BF00019521

    Article  CAS  Google Scholar 

  • Parmley SF, Smith GP (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73(2):305–318. doi:10.1016/0378-1119(88)90495-7

    Article  PubMed  CAS  Google Scholar 

  • Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9(6):582–584. doi:10.1038/nmeth.1991

    Article  PubMed  CAS  Google Scholar 

  • Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC, Leonhardt H (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3(11):887–889. doi:10.1038/nmeth953

    Article  PubMed  CAS  Google Scholar 

  • Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7(2):282–289. doi:10.1074/mcp.M700342-MCP200

    Article  PubMed  CAS  Google Scholar 

  • Saerens D, Stijlemans B, Baral TN, Nguyen Thi GT, Wernery U, Magez S, De Baetselier P, Muyldermans S, Conrath K (2008) Parallel selection of multiple anti-infectome Nanobodies without access to purified antigens. J Immunol Methods 329(1–2):138–150. doi:10.1016/j.jim.2007.10.005

    Article  PubMed  CAS  Google Scholar 

  • Sjodahl S, Rodin J, Rask L (1991) Characterization of the 12 s globulin complex of brassica-napus: evolutionary relationship to other 11–12 s storage globulins. Eur J Biochem 196(3):617–621

    Article  PubMed  CAS  Google Scholar 

  • Snapp EL (2009) Fluorescent proteins: a cell biologist’s user guide. Trends Cell Biol 19(11):649–655. doi:10.1016/j.tcb.2009.08.002

    Article  PubMed  CAS  Google Scholar 

  • Stoevesandt O, Taussig MJ (2012) European and international collaboration in affinity proteomics. New Biotechnol 29(5):511–514. doi:10.1016/j.nbt.2012.05.003

    Article  CAS  Google Scholar 

  • Tandang MRG, Atsuta N, Maruyama N, Adachi M, Utsumi S (2005) Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering. J Agric Food Chem 53(22):8736–8744. doi:10.1021/Jf050871y

    Article  PubMed  CAS  Google Scholar 

  • van der Klei H, Van Damme J, Casteels P, Krebbers E (1993) A fifth 2S albumin isoform is present in Arabidopsis thaliana. Plant Physiol 101(4):1415–1416

    Article  PubMed  Google Scholar 

  • Van Leene J, Hollunder J, Eeckhout D, Persiau G, Van De Slijke E, Stals H, Van Isterdael G, Verkest A, Neirynck S, Buffel Y, De Bodt S, Maere S, Laukens K, Pharazyn A, Ferreira PCG, Eloy N, Renne C, Meyer C, Faure J-D, Steinbrenner J, Beynon J, Larkin JC, Van de Peer Y, Hilson P, Kuiper M, De Veylder L, Van Onckelen H, Inzé D, Witters E, De Jaeger G (2010) Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana. Mol Syst Biol 6:397. doi:10.1038/msb.2010.53

    PubMed  Google Scholar 

  • Vanhecke D, Graber W, Studer D (2008) Close-to-native ultrastructural preservation by high pressure freezing. Methods Cell Biol 88:151–164. doi:10.1016/S0091-679X(08)00409-3

    Article  PubMed  Google Scholar 

  • Vanlandschoot P, Stortelers C, Beirnaert E, Ibañez LI, Schepens B, Depla E, Saelens X (2011) Nanobodies®: new ammunition to battle viruses. Antiviral Res 92(3):389–407. doi:10.1016/j.antiviral.2011.09.002

    Article  PubMed  CAS  Google Scholar 

  • Yewdell JW, Lacsina JR, Rechsteiner MC, Nicchitta CV (2011) Out with the old, in with the new? Comparing methods for measuring protein degradation. Cell Biol Int 35(5):457–462. doi:10.1042/CBI20110055

    Article  PubMed  CAS  Google Scholar 

  • Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, Stone E, Brisson J-R, MacKenzie CR (2004) Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J Mol Biol 335(1):49–56. doi:10.1016/j.jmb.2003.09.034

    Article  PubMed  CAS  Google Scholar 

  • Zhu X, Wang L, Liu R, Flutter B, Li S, Ding J, Tao H, Liu C, Sun M, Gao B (2010) COMBODY: one-domain antibody multimer with improved avidity. Immunol Cell Biol 88(6):667–675. doi:10.1038/icb.2010.21

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Geert De Jaeger and his research group for advice and help, Gholamreza Hassanzadeh Ghassabeh for helpful discussions and for providing the pHEN6c-GBP vector, Jonah Nolf and Gijs De Cort for technical assistance and Annick Bleys for help in preparing the manuscript. T.D.M. is indebted to the Agency for Innovation by Science and Technology (IWT) for a predoctoral fellowship.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Plant Systems Biology, VIB, 9052, Ghent, Belgium

    Thomas De Meyer, Dominique Eeckhout, Riet De Rycke, Sylvie De Buck & Ann Depicker

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium

    Thomas De Meyer, Dominique Eeckhout, Riet De Rycke, Sylvie De Buck & Ann Depicker

  3. Department of Structural Biology, VIB, 1050, Brussel, Belgium

    Serge Muyldermans

  4. Research Unit of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1050, Brussel, Belgium

    Serge Muyldermans

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  1. Thomas De Meyer
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  2. Dominique Eeckhout
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  3. Riet De Rycke
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  4. Sylvie De Buck
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  5. Serge Muyldermans
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  6. Ann Depicker
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Correspondence to Ann Depicker.

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De Meyer, T., Eeckhout, D., De Rycke, R. et al. Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins. Plant Mol Biol 84, 83–93 (2014). https://doi.org/10.1007/s11103-013-0118-0

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