Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription
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Date
2022
Authors
Tersteeg, Scott
Mrozowich, Tyler
Henrickson, Amy
Demeler, Borries
Patel, Trushar R.
Journal Title
Journal ISSN
Volume Title
Publisher
Canadian Science Publishing
Abstract
Inorganic 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 precip-
itating 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 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, analytical ultracentrifugation, and small-angle X-ray scattering, 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 laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly
reducing the financial strain of large-scale RNA production.
Description
Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) applies
Keywords
Inorganic pyrophosphatase , RNA in vitro transcription , PPase , Analytical ultracentrifuge
Citation
Tersteeg, S., Mrozowich, T., Henrickson, A., Demeler, B., & Patel, T. R. (2022). Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription. Biochemistry and Cell Biology, 100(5), 425-436. https://doi.org/10.1139/bcb-2022-0118