Systems and strategies for plant protein expression

Anthony J. Zmuda; Thomas D. Niehaus

doi:10.1016/bs.mie.2022.08.002

Systems and strategies for plant protein expression

Anthony J. Zmuda, Thomas D. Niehaus

Plant and Microbial Biology

Research output: Chapter in Book/Report/Conference proceeding › Chapter

3 Scopus citations

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	https://doi.org/10.1016/bs.mie.2022.08.002
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
Amino Acid Sequence
Arabidopsis/genetics
Plant Proteins/genetics
NAD/metabolism

PubMed: MeSH publication types

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10.1016/bs.mie.2022.08.002

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title = "Systems and strategies for plant protein expression",

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.",

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",

author = "Zmuda, {Anthony J.} and Niehaus, {Thomas D.}",

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: {\textcopyright} 2022 Elsevier Inc.",

year = "2023",

month = jan,

doi = "10.1016/bs.mie.2022.08.002",

language = "English (US)",

isbn = "9780443185847",

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AU - Zmuda, Anthony J.

AU - Niehaus, Thomas D.

N1 - 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.

PY - 2023/1

Y1 - 2023/1

N2 - 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.

AB - 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.

KW - Arabidopsis thaliana

KW - Escherichia coli

KW - Heterologous expression

KW - Metabolite damage

KW - Metabolite repair

KW - NAD(P)HX

KW - Plant enzyme expression

KW - Plant metabolism

KW - Recombinant protein expression

KW - Transit peptide prediction

KW - Amino Acid Sequence

KW - Arabidopsis/genetics

KW - Plant Proteins/genetics

KW - NAD/metabolism

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M3 - Chapter

C2 - 36710015

AN - SCOPUS:85138558332

SN - 9780443185847

T3 - Methods in Enzymology

SP - 3

EP - 34

BT - Biochemical Pathways and Environmental Responses in Plants

A2 - Jez, Joseph

PB - Academic Press Inc.

ER -

Systems and strategies for plant protein expression

Abstract

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Keywords

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