Abstract
Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.
Keywords: Hsp90, Cdc37, Cdk4, Chaperone, Kinase, 2A peptides, Yeast protein expression
Background
Robustly forming complexes between Hsp90 molecular chaperone and its client kinases has proven to be refractory in vitro. Previous work indicated that overexpression of Hsp90’s co-chaperone Cdc37 together with a client kinase in insect Sf9 cells led to a stable complex between Sf9 Hsp90, exogenous Cdc37 and exogenous kinase (Vaughan et al., 2006). However, insect cell culture requires special equipment, is more difficult to genetically manipulate and is significantly slower to both grow and clone into than other well studied expression systems, like bacteria and yeast. Co-expression of the above proteins in E. coli did not yield soluble kinase/stable complex. We reasoned that Saccharomyces cerevisiae would possess the necessary machinery to help fold and facilitate the complex formation and sought to generate the complex between human Hsp90 beta, human Cdc37 and human Cdk4 kinase by co-expressing these proteins in S. cerevisiae. To attain stoichiometric expression of the three proteins, we utilized viral 2A peptides, which allowed transcription of the three proteins on one mRNA with subsequent cleaving at the translation stage. This system has been utilized in human cell lines and in rabbit reticulolysates (Kim et al., 2011; Minskaia and Ryan, 2013), but to our knowledge this is the first utilization of 2A viral expression peptides in S. cerevisiae.
Materials and Reagents
Equipment
Procedure
Data analysis
Sample prepared in this way was subsequently prepared for cryo electron microscopy (cryoEM) followed by data collection and reconstruction. The protocols utilized for these steps are described in great detail in Verba et al., 2016.
Notes
Recipes
Acknowledgments
We thank Akihiko Arakawa (Yokoyama Lab) for sharing their insect cell purification protocol, Yao Fan (Arkin Lab) for the yeast expression vector, and D.A.A. lab members for helpful discussions, specifically Miguel Betegon for the suggestion of using viral 2A peptides and Michelle Moritz for yeast expression strain. Support for this work was provided by PSI-Biology grant U01 GM098254 (to D.A.A.), AACR-BCRF Grant 218084 for Translational Breast Cancer Research (to D.A.A.), The Cabala Family gift (to D.A.A.), HHMI International Student Research Fellowship (to K.V.) and the Howard Hughes Medical Institute (to D.A.A.).
References
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