title: A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.
abstract: Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
authors: Hong
authors: H
authors: Na
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: John D
authors: JD
authors: Laver
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
authors: Jouhyun
authors: J
authors: Jeon
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
authors: Fateh
authors: F
authors: Singh
authors: Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6 Department of Cell and Systems Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6.
authors: Kristin
authors: K
authors: Ancevicius
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Yujie
authors: Y
authors: Fan
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Wen Xi
authors: WX
authors: Cao
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Kun
authors: K
authors: Nie
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Zhenglin
authors: Z
authors: Yang
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Hua
authors: H
authors: Luo
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors: Miranda
authors: M
authors: Wang
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors: Olivia
authors: O
authors: Rissland
authors: Department of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6 Department of Cell and Systems Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada L5L 1C6.
authors: J Timothy
authors: JT
authors: Westwood
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Department of Computer Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Philip M
authors: PM
authors: Kim
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Craig A
authors: CA
authors: Smibert
authors: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Howard D
authors: HD
authors: Lipshitz
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada M5S 3E1 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Sachdev S
authors: SS
authors: Sidhu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D022801:Complementarity Determining Regions; D029721:Drosophila Proteins / Q000276:immunology* / Q000302:isolation & purification; D004331:Drosophila melanogaster; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012261:Ribonucleoproteins / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D000941:Antigens; D022801:Complementarity Determining Regions; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins
keywords: Fab; RNA-binding protein; phage display; ribonucleoprotein complex; synthetic antibody