Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription

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dc.contributor.authorTersteeg, Scott
dc.contributor.authorMrozowich, Tyler
dc.contributor.authorHenrickson, Amy
dc.contributor.authorDemeler, Borries
dc.contributor.authorPatel, Trushar R.
dc.date.accessioned2022-10-25T18:02:10Z
dc.date.available2022-10-25T18:02:10Z
dc.date.issued2022
dc.descriptionOpen access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesen_US
dc.description.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 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.en_US
dc.description.peer-reviewYesen_US
dc.identifier.citationTersteeg, 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-2022-0118en_US
dc.identifier.urihttps://hdl.handle.net/10133/6361
dc.language.isoen_USen_US
dc.publisherCanadian Science Publishingen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistryen_US
dc.publisher.facultyArts and Scienceen_US
dc.publisher.institutionUniversity of Lethbridgeen_US
dc.publisher.institutionUniversity of Albertaen_US
dc.publisher.institutionUniversity of Calgaryen_US
dc.publisher.urlhttps://doi.org/10.1139/bcb-2022-0118en_US
dc.subjectInorganic pyrophosphataseen_US
dc.subjectRNA in vitro transcriptionen_US
dc.subjectPPaseen_US
dc.subjectAnalytical ultracentrifugeen_US
dc.subject.lcshRNA
dc.subject.lcshCentrifuges
dc.subject.lcshSmall-angle x-ray scattering
dc.titlePurification and characterization of inorganic pyrophosphatase for in vitro RNA transcriptionen_US
dc.typeArticleen_US
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