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

摘要

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 1mg/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 ~7.5nM. 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.