Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells.
Metabotropic glutamate receptors (mGluRs) play important roles in the function and regulation of the central nervous system. Structural studies are necessary for the detailed understanding of their mechanisms of action. However, overexpression and purification of functional receptors in quantities required for these studies proves to be a major challenge. In this study we report the overexpression of a Drosophila melanogaster mGluR (DmGluRA) by using a baculovirus-insect cell expression system. Expression was tested in two different insect cell hosts (Sf9 and Hi5) and analyzed by performing expression kinetics. Pharmacological characterization of the recombinant receptor by radioactive glutamate binding assays showed a profile similar to group II mGluRs, as previously reported, when the receptor was expressed in mammalian systems. The B(max) value reached 11 pM receptor/mg Sf9-membrane protein. A monoclonal antibody against DmGluRA was generated by genetic immunization and used to purify the receptor.
Panneels
Valérie
V
Biochemie-Zentrum Heidelberg, Universität Heidelberg, INF328, 69120 Heidelberg, Germany.
Eroglu
Cagla
C
Cronet
Philippe
P
Sinning
Irmgard
I
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004331:Drosophila melanogaster; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology
D018094:Receptors, Metabotropic Glutamate; D018698:Glutamic Acid