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Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases

Authors
  • Preger, Charlotta1
  • Wigren, Edvard1
  • Ossipova, Elena1
  • Marks, Carolyn1
  • Lengqvist, Johan2
  • Hofström, Camilla3, 4
  • Andersson, Oskar3, 4
  • Jakobsson, Per-Johan1
  • Gräslund, Susanne1
  • Persson, Helena3, 4
  • 1 Structural Genomics Consortium, Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
  • 2 Pelago Bioscience AB, Solna, Sweden
  • 3 Science for Life Laboratory, Drug Discovery and Development, Stockholm, Sweden
  • 4 School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockholm, Sweden
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Aug 14, 2020
Volume
295
Issue
41
Pages
13981–13993
Identifiers
DOI: 10.1074/jbc.RA120.012893
PMID: 32817337
PMCID: PMC7549041
Source
PubMed Central
Keywords
Disciplines
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License
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Abstract

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

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