TY - JOUR
T1 - Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli
T2 - Application of a novel protein folding screen
AU - Chen, Guo Qiang
AU - Gouaux, Eric
PY - 1997/12/9
Y1 - 1997/12/9
N2 - 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.
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.
KW - AMPA receptor
KW - Fractional factorial folding screen
KW - In vitro folding
KW - Inclusion bodies
KW - Ligand-gated ion channel
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U2 - 10.1073/pnas.94.25.13431
DO - 10.1073/pnas.94.25.13431
M3 - Article
C2 - 9391042
AN - SCOPUS:0031471216
SN - 0027-8424
VL - 94
SP - 13431
EP - 13436
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 25
ER -