Generation and validation of recombinant antibodies to study tRNA synthetases in health and disease

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 non-canonical functions and several of them have been linked to autoimmune diseases, cancer and neurological disorders. In order to study these proteins further, recombinant high-affinity antibodies have been generated to a selection 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 (scFv) 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 mass spectrometry (IP-MS) for their ability to capture the endogenous protein from mammalian cell lysates. Interestingly, for antibodies targeting individual members of the multi-tRNA synthetase complex (MSC), we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a sub-set of binders for each target was also confirmed in immunofluorescence. To conclude, we have created an open source resource, in the form of high quality recombinant proteins and antibodies to accelerate and empower future research of the role of aaRSs in health and disease.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Susanne Gräslund  

LAB HEAD: Susanne Gräslund

PROVIDER: PXD020104 | Pride | 2020-08-13

REPOSITORIES: Pride