Expression and purification of metabotropic glutamate receptor 7 peptides.
Metabotropic glutamate receptors (mGluRs) influence a variety of second-messenger systems and ion channels. The C-terminal region of group III mGluRs interacts with the Ca(2+)-binding protein calmodulin (CaM). We intend to study the interaction between Ca(2+)/CaM and the CaM-binding motifs within mGluR(7), which is a group III mGluR. We established a recombinant protein expression and purification system for nuclear magnetic resonance (NMR) analysis of mGluR(7) peptides using Escherichia coli. Peptides of mGluR(7) conjugated to an affinity tag sequence were constructed, and protocols for expression and purification were optimized. To suppress non-specific enzymatic cleavage, the mGluR(7) fusion peptide was bound to Ca(2+)/CaM before enterokinase cleavage. This complex method for precise enzymatic reactions may be applicable for the recombinant preparation of a wide variety of peptides.
Isozumi
Noriyoshi
N
Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Ohki
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D002147:Calmodulin; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; C102719:metabotropic glutamate receptor 7; D002118:Calcium