Human cytosolic phenylalanyl-tRNA synthetase (FARS) is an enzyme that catalyzes the aminoacylation of phenylalanyl-tRNA and plays a crucial role in translation. In addition to its aminoacylation function, several non-canonical functions of FARS have been described in recent years. The presence of the FARSA and FARSB subunits in the cell nucleus has also been detected in some cell lines. The mechanisms of their nuclear transport remain poorly understood; therefore, the aim of our research was to construct mammalian expression vectors encoding karyopherins-α (KPNA1–KPNA7), karyopherin-β1 (KPNB1), and transportin-1 (TNPO1), and to investigate potential interactions between FARS subunits and nuclear transport receptors using the GST pull-down assay.
The coding sequences of the karyopherins were successfully transferred from the bacterial expression vector pGEX2TK into the mammalian expression vector pcDNA3.1 using IVA. The successful generation of the constructs was confirmed by restriction analysis, PCR, and determination of the nucleotide sequences. Recombinant proteins GST-FARSA and GST-FARSB were expressed and then purified using a bacterial expression system. Properties of the purified proteins were evaluated using dynamic light scattering (DLS) and nano differential scanning fluorimetry (nanoDSF). The DLS results showed the presence of aggregation of the recombinant proteins.
Using the GST pull-down assay, interactions between GST-FARSA and the karyopherins KPNA4, KPNA5, and KPNA6 were investigated. The performed experiments did not provide definite evidence for specific interactions between the analyzed proteins; therefore, the hypothesis of FARS nuclear transport mediated by the investigated karyopherins could not be confirmed based on the obtained results. Nevertheless, a set of mammalian expression constructs was successfully generated, providing an important foundation for further studies on FARS nuclear transport and its potential non-canonical functions.
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