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  Purification of crystallization-grade RNA polymerase I from S. cerevisiae.

Engel, C. (2016). Purification of crystallization-grade RNA polymerase I from S. cerevisiae. In A. Németh (Ed.), The Nucleolus (pp. 85-97). New York, N.Y.: Springer; Humana Pr. doi:10.1007/978-1-4939-3792-9_7.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-43D4-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-A4DB-2
Genre: Book Chapter

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2341880.pdf (Publisher version), 523KB
 
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 Creators:
Engel, C.1, Author              
Affiliations:
1Department of Molecular Biology, MPI for Biophysical Chemistry, Max Planck Society, ou_1863498              

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Free keywords: Protein purification; RNA polymerase I; Saccharomyces cerevisiae fermentation; Transcription; Affinity and size exclusion chromatography
 Abstract: Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography.

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Language(s): eng - English
 Dates: 2016-01-012016-08-31
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1007/978-1-4939-3792-9_7
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Title: The Nucleolus
Source Genre: Book
 Creator(s):
Németh, A., Editor
Affiliations:
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Publ. Info: New York, N.Y. : Springer; Humana Pr.
Pages: XI, 279 Volume / Issue: - Sequence Number: - Start / End Page: 85 - 97 Identifier: ISBN: 978-1-4939-3790-5

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Title: Methods in Molecular Biology
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Pages: - Volume / Issue: 1455 Sequence Number: - Start / End Page: - Identifier: ISSN: 1064-3745