High Throughput Screening of Purified Proteins for Enzymatic Activity

Understanding the functions of every protein in the proteome is one of the great challenges of the postgenomic era. Global genome sequencing efforts revealed that in any genome 30–50% of genes encode proteins with unknown function (hypothetical proteins). To directly test purified hypothetical proteins for catalytic activity, the authors have designed a series of general and specific enzymatic screens. The described screens are designed to detect hydrolases (phosphatases, phosphodiesterases, proteases, and esterases), and oxidore-ductases (dehydrogenases and oxidases). The general screens use either general chromogenic substrates or pools of substrates. The positive hits with the model substrates are then tested in the secondary screens with a set of potential natural substrates, or the substrate pools can be deconvoluted to identify the preferred in vitro substrate. The identification of a biochemical activity of a hypothetical protein helps to determine its cellular role.