Purification and Characterization of Inorganic Pyrophosphatase for in vitro RNA Transcription

Abstract

AbstractInorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and of high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering (SEC-MALS-DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for labs involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.Statement of SignificanceWe show an easy two-step purification procedure to efficiently produce large quantities of iPPase, an expensive component of IVT reactions. Laboratory-produced iPPase can significantly reduce the financial strain on research labs that rely on large-scale RNA production for experiments. Furthermore, we show for the first time, using a combination of orthogonal biophysical techniques, that yeast iPPase assembles into higher-order oligomers similar to bacterial iPPase.