Characterization of Prolyl-4-Hydroxylase Substrate Specificity Using Pichia pastoris as an Efficient Eukaryotic Expression System.
| Autor: | Elsäßer G; Department of Plant Physiology and Biochemistry, Institute of Biology, University of Hohenheim, Stuttgart, Germany., Seidl T; Department of Plant Physiology and Biochemistry, Institute of Biology, University of Hohenheim, Stuttgart, Germany., Pfannstiel J; Core Facility Hohenheim, Mass Spectrometry Module, University of Hohenheim, Stuttgart, Germany., Schaller A; Department of Plant Physiology and Biochemistry, Institute of Biology, University of Hohenheim, Stuttgart, Germany., Stührwohldt N; Department of Plant Physiology and Biochemistry, Institute of Biology, University of Hohenheim, Stuttgart, Germany. nils.stuehrwohldt@uni-hohenheim.de. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2731, pp. 59-80. |
| DOI: | 10.1007/978-1-0716-3511-7_5 |
| Abstrakt: | The use of eukaryotic expression systems facilitates the heterologous expression of complex eukaryotic proteins in their post-translationally modified and biologically active state, as a prerequisite for subsequent biochemical characterization and functional analysis. Here we describe the complete workflow for the expression of Arabidopsis thaliana prolyl-4-hydroxylases (P4Hs) in the methylotrophic yeast Pichia pastoris (renamed as Komagataella phaffii), for the extraction of the recombinant enzymes, purification by affinity chromatography, and characterization of P4H activity and specificity toward oligopeptide substrates by mass spectrometry. We expressed eight of the 13 Arabidopsis P4Hs and show that they are all active against proline-rich extensin-derived peptides. However, three of them differed in substrate specificity and were also able to hydroxylate the CLEL9 signaling peptide, featuring a single proline within its mature peptide sequence. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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