Systems and strategies for plant protein expression

Anthony J. Zmuda, Thomas D. Niehaus

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

At least a quarter of the protein-encoding genes in plant genomes are predicted to encode enzymes for which no physiological function is known. Determining functions for these uncharacterized enzymes is key to understanding plant metabolism. Functional characterization typically requires expression and purification of recombinant enzymes to be used in enzyme assays and/or for protein structure elucidation studies. Here, we describe several practical considerations used to improve the heterologous expression and purification of Arabidopsis thaliana and Zea mays NAD(P)HX dehydratase (NAXD) and NAD(P)HX epimerase (NAXE), two enzymes that are involved in repair of chemically damaged NAD(P)H cofactors. We provide protocols for transit peptide prediction and construct design, expression in Escherichia coli, and purification of NAXD and NAXE. Many of these strategies are generally applicable to the purification of any plant protein.

Original languageEnglish (US)
Title of host publicationBiochemical Pathways and Environmental Responses in Plants
Subtitle of host publicationPart B
EditorsJoseph Jez
PublisherAcademic Press Inc.
Pages3-34
Number of pages32
ISBN (Print)9780443185847
DOIs
StatePublished - Jan 2023

Publication series

NameMethods in Enzymology
Volume680
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
Much of the work described in here was performed under the supervision and in the laboratory of Andrew D. Hanson, Horticultural Sciences Department, University of Florida.

Publisher Copyright:
© 2022 Elsevier Inc.

Keywords

  • Arabidopsis thaliana
  • Escherichia coli
  • Heterologous expression
  • Metabolite damage
  • Metabolite repair
  • NAD(P)HX
  • Plant enzyme expression
  • Plant metabolism
  • Recombinant protein expression
  • Transit peptide prediction
  • Amino Acid Sequence
  • Arabidopsis/genetics
  • Plant Proteins/genetics
  • NAD/metabolism

PubMed: MeSH publication types

  • Journal Article

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