Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: Preparation for a functional analysis

Ahmed Afzal, David A. Lightfoot

Research output: Contribution to journalArticle

Abstract

Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0 M urea in 0.5 M increments. Purified RHG1 protein obtained from the 1.5 and 7.0 M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0 M urea sample, whereas refolding from the 1.5 M urea sample yielded 0.2 mg/ml protein. The 7.0 M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.

Original languageEnglish (US)
Pages (from-to)346-355
Number of pages10
JournalProtein Expression and Purification
Volume53
Issue number2
DOIs
StatePublished - Jun 1 2007

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Functional analysis
Disease Resistance
Inclusion Bodies
Soybeans
Escherichia coli
Urea
Protein Refolding
Proteins
Circular Dichroism
Thermodynamics
Plant Proteins
Glutathione Disulfide
Leucine
Circular dichroism spectroscopy
Glutathione
Arginine
Spectrum Analysis
Kinetics
Phosphotransferases
Dichroism

Keywords

  • Expression in E. coli
  • His-tag purification
  • Plant disease resistance
  • Protein folding
  • SCN

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: Preparation for a functional analysis",
abstract = "Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0 M urea in 0.5 M increments. Purified RHG1 protein obtained from the 1.5 and 7.0 M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0 M urea sample, whereas refolding from the 1.5 M urea sample yielded 0.2 mg/ml protein. The 7.0 M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.",
keywords = "Expression in E. coli, His-tag purification, Plant disease resistance, Protein folding, SCN",
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T1 - Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies

T2 - Preparation for a functional analysis

AU - Afzal, Ahmed

AU - Lightfoot, David A.

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N2 - Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0 M urea in 0.5 M increments. Purified RHG1 protein obtained from the 1.5 and 7.0 M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0 M urea sample, whereas refolding from the 1.5 M urea sample yielded 0.2 mg/ml protein. The 7.0 M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.

AB - Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0 M urea in 0.5 M increments. Purified RHG1 protein obtained from the 1.5 and 7.0 M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0 M urea sample, whereas refolding from the 1.5 M urea sample yielded 0.2 mg/ml protein. The 7.0 M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.

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