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Expression and Purification of Human Mitochondrial Intramembrane Protease PARL

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Structure and Function of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2302))

Abstract

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1 mg of pure PARL per liter of yeast culture.

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Author information

Author notes

  1. Cory Brooks

    Present address: Department of Chemistry, California State University, Fresno, CA, USA

Authors and Affiliations

  1. Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada

    Elena Arutyunova, Laine Lysyk, Melissa Morrison, Cory Brooks & M. Joanne Lemieux

Authors
  1. Elena Arutyunova
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  2. Laine Lysyk
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  3. Melissa Morrison
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  4. Cory Brooks
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  5. M. Joanne Lemieux
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Corresponding author

Correspondence to M. Joanne Lemieux .

Editor information

Editors and Affiliations

  1. School of Biological Sciences and School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Ingeborg Schmidt-Krey

  2. School of Physics, Georgia Institute of Technology, Atlanta, GA, USA

    James C. Gumbart

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Arutyunova, E., Lysyk, L., Morrison, M., Brooks, C., Joanne Lemieux, M. (2021). Expression and Purification of Human Mitochondrial Intramembrane Protease PARL. In: Schmidt-Krey, I., Gumbart, J.C. (eds) Structure and Function of Membrane Proteins. Methods in Molecular Biology, vol 2302. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1394-8_1

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  • DOI: https://doi.org/10.1007/978-1-0716-1394-8_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1393-1

  • Online ISBN: 978-1-0716-1394-8

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