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Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro

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Plant and Food Carotenoids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2083))

Abstract

Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires 14C-labeled substrate and allows to determine its incorporation into phytoene as well as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase homologs from various plant species.

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Acknowledgments

This work was supported in part by the HarvestPlus research consortium (grant 2014H6320.FRE) to R.W. The authors thank the former members of the laboratory of Prof. Peter Beyer for their contributions to establishing this method.

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Author notes

  1. Maurizio Camagna

    Present address: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Authors and Affiliations

  1. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Maurizio Camagna

  2. Institute for Biology II, University of Freiburg, Freiburg, Germany

    Ralf Welsch

Authors
  1. Maurizio Camagna
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  2. Ralf Welsch
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Corresponding author

Correspondence to Ralf Welsch .

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Editors and Affiliations

  1. CSIC-IRTA-UAB-UB, Centre for Research in Agricultural Genomics (CRAG), Barcelona, Spain

    Manuel Rodríguez-Concepción

  2. Institute for Biology II, University of Freiburg, Freiburg, Germany

    Ralf Welsch

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Camagna, M., Welsch, R. (2020). Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro. In: Rodríguez-Concepción, M., Welsch, R. (eds) Plant and Food Carotenoids. Methods in Molecular Biology, vol 2083. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9952-1_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9952-1_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9951-4

  • Online ISBN: 978-1-4939-9952-1

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