Prosequence switching: An effective strategy to produce biologically activeE. coliheat-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.

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif

“…Accordingly, and to alleviate the cost of production of labeled peptides, additional purification strategies of peptides for NMR studies have been reported using His [], maltose‐binding protein , the RNA‐binding domain of human hnRNCP1 , GB1 domain , ketosteroid isomerase , thioredoxin , and even a combination of tags yielding high‐resolution spectra. Recently, a protocol for the purification of the cysteine‐rich heat‐stable enterotoxin was developed, in which its three disulfide bonds and proper folding were preserved during purification by the use of human uroguanylin as a tag (Table ) as demonstrated by the high‐resolution HSQC spectrum of the peptide . Protease cleavage often triggers peptide proteolysis and chemical cleavage, such as with cyanogen bromide, and is an alternative option to isolating untagged peptides .…”

Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins

“…To promote disulfide bridge formation, this approach used the E. coli Origami B (DE3) strain, which has an oxidative cytoplasm. By further using a protocol requiring two enzymatic cleavage steps, biologically active ST peptide from the fusion protein was obtained [ 35 ].…”