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

Preger C, Wigren E, Ossipova E, Marks C, Lengqvist J, Hofström C, Andersson O, Jakobsson P, Gräslund S, Persson H

J. Biol. Chem. 295 (41) 13981-13993 [2020-10-09; online 2020-08-14]

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.

Drug Discovery and Development (DDD) [Collaborative]

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PubMed 32817337

DOI 10.1074/jbc.RA120.012893

Crossref 10.1074/jbc.RA120.012893

pii: RA120.012893
pmc: PMC7549041