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High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae

  • Jennifer M. Johnson
  • Franklin A. HaysEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Yeast expression Protein expression Protein purification Fermentation Membrane protein 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Stephenson Cancer CenterUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  3. 3.Harold Hamm Diabetes CenterUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA

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