Establishing the history of safe use of the OsDIAT protein: preliminary characterization of the recombinant protein and in silico analyses

The drought-induced branched-chain amino acid aminotransferase (OsDIAT) from Oryza sativa belongs to a protein family with a long history of safe use (HOSU). In this study, we conducted a preliminary characterization of the recombinant OsDIAT protein produced in Escherichia coli, together with in silico and literature-based analyses, to contribute to the weight of evidence supporting the HOSU of the OsDIAT protein. In simulated gastric fluid (SGF), the intact recombinant protein was no longer detectable within 1 min, whereas it remained detectable in simulated intestinal fluid (SIF) for 60 min in a separate time-course assay; sequential SGF–SIF digestion was not performed. When heated at 95 °C (the only temperature tested), the intact protein band was no longer detectable from 60 min onward; enzymatic activity was not measured. In silico analyses identified no significant sequence similarity to known proteinaceous toxins or allergens, and domain analysis was consistent with a pyridoxal phosphate-dependent aminotransferase. Two potential N-glycosylation sites were predicted, but because no signal peptide or transmembrane domain was predicted, the protein is not expected to enter the secretory pathway, and the sites are unlikely to be modified in the native protein. These preliminary data add to the weight of evidence supporting the HOSU of the OsDIAT protein. However, they relate only to the microbially produced recombinant protein, and further studies, including demonstration of equivalence with the plant-expressed protein, are required before these findings can be associated with any genetically modified plant expressing this protein.