Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae
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 . 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.
Key wordsProtein purification RNA polymerase I Transcription Saccharomyces cerevisiae fermentation Affinity and size exclusion chromatography
I thank Patrick Cramer for supervising and supporting all research and method development. I acknowledge the contribution of all past and present members of the Pol I subgroup to a constant evolution of the protocol, especially Claus Kuhn and Stefan Jennebach. Thanks also to Carlo Bäjen, Tobias Gubbey, Sarah Sainsbury, Andrea Stutz and Youwei Xu. This work was funded by the Boehringer Ingelheim Fonds and the Max Planck Society.
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