PROCESSING AND CHARACTERIZATION OF HUMAN PROGUANYLIN EXPRESSED IN ESCHERICHIA-COLI

Guanylin is a 15-amino acid peptide hormone that was originally isolat ed from the jejunum of the rat small intestine and shown to be an endo genous activator of the intestinal heat-stable enterotoxin receptor-gu anylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone , proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processin g of proguanylin in vitro, we have generated large quantities of the p roperly folded prohormone by constructing an expression vector that di rects its secretion into the periplasmic space of Escherichia coli. Th e bacterially expressed human proguanylin was then processed to smalle r C-terminal fragments by protease digestion. Digestion with trypsin o r lysine-C generated C-terminal peptides of different length, which ha ve been purified and characterized. Guanylin-22 and guanylin-32 have b inding affinities and biological activities similar to guanylin-15, wh ile guanylin-63 and the entire proguanylin have only minimal bioactivi ty. Circular dichroism spectroscopy reveals that proguanylin is a stab ly folded protein containing mostly beta-sheet and beta-turn structure .