Purification and validation of asparaginyl-tRNA synthetase heterodimer with indistinguishable subunits

Oligomerization can influence the stability and activity of a protein. The majority of enzymes, including aminoacyl-tRNA synthetases, become catalytically active upon forming homodimers. Residues located at the dimerization interface are highly conserved and mutations arising within can cause severe disease phenotypes. Beyond homozygous mutations, other disease-causing mutations, such as compound heterozygous and mono-allelic mutations, can lead to the formation of heterodimers between two distinct subunits. Purifying a recombinant heterodimer is required for its thorough characterization in vitro, yet there is a lack of established biochemical methods for the preparation. Here we describe a heterodimer purification and validation method with the example of a disease-causing mono-allelic, nonsense mutation R534* in cytoplasmic asparaginyl-tRNA synthetase (NARS1 or AsnRS). Our method involves co-expression of two separately tagged constructs to allow for purification of the wild-type and the R534* mutant heterodimers. While the two subunits can hardly be distinguished by size, their separate detection is achieved by western blotting against the tags. Quantification analysis confirmed that the subunits within the heterodimer are present in nearly equal proportions. This simple protocol can be adapted to study other size-indistinguishable heterodimers.