Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins

Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Generation of VHH antibodies against the Arabidopsis thaliana seed storage proteins

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Publisher
Springer Netherlands
Copyright
Copyright © 2013 by Springer Science+Business Media Dordrecht
Subject
Life Sciences; Plant Sciences; Biochemistry, general; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-013-0118-0
Publisher site
See Article on Publisher Site

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.

Journal

Plant Molecular BiologySpringer Journals

Published: Aug 21, 2013

References

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    Arbabi-Ghahroudi, M; Tanha, J; MacKenzie, R
  • High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy
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  • Expression and processing of an Arabidopsis 2S albumin in transgenic tobacco
    Clercq, A; Vandewiele, M; Rycke, R; Damme, J; Montagu, M; Krebbers, E; Vandekerckhove, J
  • Generating recombinant antibodies to the complete human proteome
    Dübel, S; Stoevesandt, O; Taussig, MJ; Hust, M
  • Single-domain antibody fragments with high conformational stability
    Dumoulin, M; Conrath, K; Meirhaeghe, A; Meersman, F; Heremans, K; Frenken, LG; Muyldermans, S; Wyns, L; Matagne, A
  • A technology platform for the fast production of monoclonal recombinant antibodies against plant proteins and peptides
    Eeckhout, D; Clercq, A; Slijke, E; Leene, J; Stals, H; Casteels, P; Persiau, G; Vercammen, D; Breusegem, F; Zabeau, M; Inzé, D; Jespers, L; Depicker, A; Jaeger, G
  • Protein structure modeling indicates hexahistidine-tag interference with enzyme activity
    Freydank, A-C; Brandt, W; Dräger, B

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