4.3 Article

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription

期刊

BIOCHEMISTRY AND CELL BIOLOGY
卷 100, 期 5, 页码 425-436

出版社

CANADIAN SCIENCE PUBLISHING
DOI: 10.1139/bcb-2022-0118

关键词

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

资金

  1. NSERC PGS-D fellowship
  2. NSERC CGS
  3. DIAMOND Light Source B21 beamline
  4. [SM26855]

向作者/读者索取更多资源

This study demonstrates that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility, and laboratory-purified iPPase is as effective as commercially available iPPase. Our work provides a robust protocol for efficiently producing active iPPase, significantly reducing the financial strain of large-scale RNA production.
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 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 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.

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