Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: application of a novel protein folding screen.
Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. & Keinänen, K. (1995) EMBO J. 14, 6327-6332] of the rat alpha-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 alpha-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.
Chen
G Q
GQ
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Gouaux
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D014357:Trypsin; D013917:Thrombin; C104722:glutamate receptor ionotropic, AMPA 2