title: A new method for establishing stable cell lines and its use for large-scale production of human guanylyl cyclase-B receptor and of the extracellular domain.
abstract: Guanylyl cyclase-B receptor (GC-B) is a membrane receptor that induces intracellular accumulation of cGMP when a specific ligand, C-type natriuretic peptide (CNP), binds to the extracellular ligand-binding domain (ECD). Despite of its medical and biological importance, characterization of GC-B is hampered by limited amounts of protein obtainable. To circumvent this problem, a method was developed for rapidly and semi-automatically establishing stable cell lines specialized for large-scale production. This method, utilizing a bicistronic expression vector for co-expressing a green fluorescent protein and FACS-based selection of high-expressing cells, is generally applicable. It worked particularly well with the ECD and yielded highly purified ECD at 1 mg/l of culture medium by affinity chromatography using modified CNPs. Measurements of ligand-binding and guanylyl cyclase activities for various natriuretic peptides showed that, as expected, CNP is by far the most potent agonist of GC-B with IC(50) of ~7.5 nM. This value is at least an order of magnitude larger than that reported earlier but similar to that established with the guanylyl cyclase-A receptor for its ligand, atrial natriuretic peptide. The methods developed here will be useful, at the least, for characterizing other members of the guanylyl cyclase receptor family.
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okamoto
authors.forename: Haruo
authors.initials: H
authors.lastname: Ogawa
authors.forename: Chikashi
authors.initials: C
authors.lastname: Toyoshima
mesh_terms: D000595:Amino Acid Sequence; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000099242:Atrial Natriuretic Factor Receptor B
chemical_list: D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B