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High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae

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High-Throughput Protein Production and Purification

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2025))

Abstract

This chapter outlines a protocol to assess viability for large-scale protein production and purification for selected targets from an initial medium-throughput cloning strategy. Thus, one can assess a broad number of potential candidate proteins, mutants, or expression variants using an empirically minimalistic approach. In addition, a key output from this protocol is utilization of Saccharomyces cerevisiae as a means for the efficient screening and production of purified proteins. The primary focus in this protocol is overexpression of polytopic integral membrane proteins though methods can be equally applied to soluble proteins. The protocol starts with outlining high-throughput (sans robotics) cloning of expression proteins into a dual-tag yeast expression plasmid. These membrane proteins are then screened for expression level, detergent solubilization, initial purity, and chromatography characteristics. Both small- and large-scale expression methods are discussed along with fermentation.

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Acknowledgments

The authors would like to acknowledge Jennifer Washburn, Zygy Roe-Žurž, Hannah Schmitz, and Dr. Robert M. Stroud for their support in previous development and implementation of methods outlined in this chapter. The Hays lab is supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number R01GM118599.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Jennifer M. Johnson & Franklin A. Hays

  2. Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Franklin A. Hays

  3. Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Franklin A. Hays

Authors
  1. Jennifer M. Johnson
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  2. Franklin A. Hays
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Correspondence to Franklin A. Hays .

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Editors and Affiliations

  1. Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille cedex 9, France

    Renaud Vincentelli

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Johnson, J.M., Hays, F.A. (2019). High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9624-7_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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