Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding.
An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies.
Kiefer
H
H
Stockholm University, Department of Biochemistry, Sweden.
Krieger
J
J
Olszewski
J D
JD
Von Heijne
G
G
Prestwich
G D
GD
Breer
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D005982:Glutathione Transferase; D008961:Models, Structural; D009812:Odorants; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D018035:Receptors, Odorant / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016324:Sequence Tagged Sites
D011956:Receptors, Cell Surface; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase