Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription

Scott Tersteeg, Tyler Mrozowich, Amy Henrickson, Borries Demeler, Trushar R. Patel

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)425-436
Number of pages12
JournalBiochemistry and Cell Biology
Volume100
Issue number5
DOIs
StatePublished - Oct 2022
Externally publishedYes

Keywords

  • RNA in vitro transcription
  • analytical ultracentrifuge
  • iPPase
  • inorganic pyrophosphatase
  • small-angle X-ray scattering

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription'. Together they form a unique fingerprint.

Cite this