A UPLC-based activity assay method for aminoglycoside phosphotransferase 4-Ia (APH4): A Selectable marker of plant...

The aminoglycoside phosphotransferase 4-Ia (APH4) is a hygromycin B phosphotransferase and catalyzes the phosphorylation of the 4-hydroxyl group of the antibiotic hygromycin B, inactivating its antibiotic activity. Therefore, APH4 has utility as a selectable marker for transformation of many plant species. However, suitable methods to measure and quantify plants expressing APH4 enzymes are lacking because of technical challenges, associated with both specificity and sensitivity. Here we describe a highly specific and sensitive APH4 enzymatic activity assay by measuring the production of adenosine 5'-diphosphate (ADP) using ultra performance liquid chromatography (UPLC)-based method. The present assay enabled determination of enzymatic activity of APH4 over a concentration range from 0.05 to 0.8 μg APH4/ml. Method performance validation sample were examined at five concentrations in triplicate in six independent sessions. Average CVs of 5.1 % for intra-assay and 11.7 % for inter-assay over all samples were determined for precision, and 6.0 % was determined for accuracy. Additionally, the method was validated for use to determine APH4 enzymatic activity in both microbially produced and plant-derived samples.