Abstract
The chapter describes the purification of enterotoxigenic Escherichia coli (ETEC) enterotoxin ST a , which is the heat-stable mouse-active enterotoxin. ETEC may induce clinically significant intestinal fluid secretion (i.e., diarrhea) in humans and animals by production of enterotoxins. Several types of ETEC enterotoxins are recognized, each with a unique structure and mechanism of action. ST a activates particulate guanylate cyclase in intestinal epithelial cells. A modification suckling mouse assay (SMA) is used for detection and quantitation of ST a throughout the purification steps. Sequence analysis of ST a peptides is performed by the manual microsequencing technique of Chang using the double-coupling method with dimethylaminoazobenze isothiocyanate/phenyl isothiocyanate. After each cycle of derivatization, the labeled amino-terminal amino acid is cleaved. For preparative purposes the described scheme can be scaled upward to produce larger amounts of ST a . The derivatized amino acids are identified by their retention times in comparison with amino acid standards derivatized and chromatographed under the same conditions.
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