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Expression and characerization of human kainate receptor subunits in Escherichia coli and mammalian cells
Author(s) -
Foldes Robert L.,
Fantaske Robert P.,
Korczak Bozena,
Hoo Ken H.,
Nutt Stephen L.,
Kamboj Rajender K.
Publication year - 1995
Publication title -
drug development research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.582
H-Index - 60
eISSN - 1098-2299
pISSN - 0272-4391
DOI - 10.1002/ddr.430340303
Subject(s) - chinese hamster ovary cell , hek 293 cells , microbiology and biotechnology , biology , cell culture , receptor , polyclonal antibodies , kainate receptor , antibody , biochemistry , glutamate receptor , genetics , ampa receptor
Cellular expression systems for human kainate receptor subunits (EAA1 and EAA2) have been developed as tools to support drug screening and rational drug design. EAA1 and EAA2 sequence‐specific polyclonal antibodies were generated to characterize polypeptide expression on introduction of appropriate plasmid expression constructs to Escherichia coli ( E. Coli ), chinese hamster ovary (CHO) and human embryonic kidney (HEK‐293) cells. A polypeptide with an apparent molecular mass of ∼ 120 kilodaltons (kDa) was identified by Western blot analysis in CHO and HEK‐293 cells expressing EAA2. Three major immunoreactive bands of 116, 110, and 90 kDa were identified in HEK‐293 cells expressing EAA1. The polyclonal antibodies will allow the direct determination of EAA1 and EAA2 expression in human brain. Pharmacological characterization of a stable CHO cell line expressing EAA2 revealed a dissociation constant for kainate (K D ) of 1.92 ± 0.28 nM (n = 3). This is the first report describing a stable cell line expressing EAA2 and the third report describing a stable cell line expressing a human glutamate receptor subunit. These studies are an important prelude to discovery of EAA1 and/or EAA2 specific drugs. © 1995 Wiley‐Liss, Inc.