Processing and characterization of human proguanylin expressed in Escherichia coli.
Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl 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 processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.
Garcia
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KC
Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080.
de Sauvage
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FJ
Struble
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Henzel
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Reilly
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Goeddel
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli; D005768:Gastrointestinal Hormones; D015870:Gene Expression; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D014357:Trypsin
D018076:DNA, Complementary; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C072685:guanylin; D014357:Trypsin; D006162:Guanylate Cyclase; D006152:Cyclic GMP