EXPRESSION OF AN OLFACTORY RECEPTOR IN ESCHERICHIA-COLI - PURIFICATION, RECONSTITUTION, AND LIGAND-BINDING

An olfactory receptor has been expressed in bacterial cells as a fusio n protein with glutathione S-transferase (GST). Overexpression of rece ptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or w ith mutants carrying three positively charged residues in the first in tracellular loop. The overexpressed fusion protein accumulated in incl usion 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 purif ied receptor was reconstituted into lipid vesicles and specific bindin g of odor ligands was shown by photoaffinity labeling and tryptophan f luorescence measurements. Thus, for the first time, an odorant recepto r/ligand pair becomes available in large amounts for biophysical and s creening studies.