Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: Application of a novel protein folding screen

Guo Qiang Chen, Eric Gouaux

Research output: Contribution to journalArticle

141 Citations (Scopus)

Abstract

Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. and Keinanen, K. (1995) EMBO J. 14, 6327-6332] of the rat α-amino-3- hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of > 100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain. Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.

Original languageEnglish (US)
Pages (from-to)13431-13436
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number25
DOIs
StatePublished - Dec 9 1997
Externally publishedYes

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Protein Folding
Glutamate Receptors
Histidine
Escherichia coli
Ligands
Proteins
Inclusion Bodies
Protein Binding
Water
bucide
propionic acid

Keywords

  • AMPA receptor
  • Fractional factorial folding screen
  • In vitro folding
  • Inclusion bodies
  • Ligand-gated ion channel

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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abstract = "Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. and Keinanen, K. (1995) EMBO J. 14, 6327-6332] of the rat α-amino-3- hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of > 100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain. Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.",
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AB - Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. and Keinanen, K. (1995) EMBO J. 14, 6327-6332] of the rat α-amino-3- hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of > 100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain. Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.

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