title: Prosequence switching: an effective strategy to produce biologically active E. coli heat-stable enterotoxin STh.
abstract: Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.
authors: a Lehrstuhl Biopolymere & Research Center for Bio-Macromolecules , Universität Bayreuth , Bayreuth 95447 , Germany .
authors: Philipp R
authors: PR
authors: Weiglmeier
authors: Hanna
authors: H
authors: Berkner
authors: Angela
authors: A
authors: Seebahn
authors: Nico
authors: N
authors: Vogel
authors: Rainer
authors: R
authors: Schreiber
authors: Birgitta M
authors: BM
authors: Wöhrl
authors: Stephan
authors: S
authors: Schwarzinger
authors: Paul
authors: P
authors: Rösch
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D001709:Biotechnology / Q000379:methods*; D002846:Chromatography, Affinity; D004768:Enterotoxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli)
keywords: heat-stable enterotoxin STh; prosequence-assisted folding; uroguanylin