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 language | English (US) |
|---|---|
| Title of host publication | Biochemical Pathways and Environmental Responses in Plants |
| Subtitle of host publication | Part B |
| Editors | Joseph Jez |
| Publisher | Academic Press Inc. |
| Pages | 3-34 |
| Number of pages | 32 |
| ISBN (Print) | 9780443185847 |
| DOIs | |
| State | Published - Jan 2023 |
Publication series
| Name | Methods in Enzymology |
|---|---|
| Volume | 680 |
| 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
PubMed: MeSH publication types
- Journal Article